Gluconobacter oxydans 2-ketoreductase enzyme and applications thereof

ABSTRACT

This invention relates to an novel  Gluconobacter oxydans  2-ketoreductase useful for the synthesis of chiral alcohols. Also related are isolated nucleic acids encoding  G. oxydans  2-ketoreductase enzymes, and enzyme fragments and variants thereof, as well as vectors and host cells comprising these nucleic acids. Further related are isolated  G. oxydans  2-ketoreductase polypeptides, and fragments and variants thereof, and antibodies that specifically bind to  G. oxydans  2-ketoreductase polypeptides, fragments, or variants. The invention also relates to methods of obtaining isolated  G. oxydans  2-ketoreductase nucleic acids, polypeptides, and antibodies, and methods of using  G. oxydans  2-ketoreductase in various reactions for industrial or pharmaceutical applications.

This invention claims priority from provisional U.S. application Ser.No. 60/341,933 filed Dec. 19, 2001, which is incorporated herein byreference in its entirety.

FIELD OF THE INVENTION

This invention relates to a novel 2-ketoreductase isolated fromGluconobacter oxydans which catalyzes the reduction of 2-pentanone toS-(+)-2-pentanol. The invention also relates to isolated nucleic acidscomprising nucleotide sequences that encode G. oxydans 2-ketoreductasepolypeptides. Also related are vectors and host cells comprising thesenucleic acids, isolated G. oxydans 2-ketoreductase polypeptides (e.g.,recombinant polypeptides), and antibodies that specifically bind to G.oxydans 2-ketoreductase polypeptides. The invention further relates tomethods of obtaining isolated G. oxydans 2-ketoreductase nucleic acids,polypeptides, and antibodies, and methods of using G. oxydans2-ketoreductase in reactions required for the synthesis of industrial orpharmaceutical compounds.

BACKGROUND OF THE INVENTION

The stereospecific reduction of carbonyl groups can be used to producechiral alcohols. Enantiomerically homogeneous chiral secondary alcoholsare useful intermediates for pharmaceuticals, and may be prepared fromketones using NADH/NADPH-dependent secondary alcohol dehydrogenases.Several biochemical and chemical approaches have been employed in thesynthesis of enantiomerically pure alcohols. These approaches includestereospecific chemical reduction of ketones, enzymatic hydrolysis ofracemic esters, and enzymatic esterification of racemic alcohols.Notably, microbial enzymes have been used for the synthesis of chiralalcohols at laboratory, pilot, and production scale (C. J. Sih and C.-S.Chen, 1984, Angew Chem. Int. Ed. Engl. 23:570-578; O. P. Ward and C. S.Young, 1990, Enzyme Microb. Technol. 12:482-493). These synthesisreactions are typically carried out using resting cells, isolatedenzymes, and/or cloned and overexpressed enzymes.

In particular, Rhodococcus erythropolis NADH-dependent carbonylreductase has been used with a wide range of substrates, including2-ketones, 3-ketones, keto-esters, and aromatic ketones (T. Zelinski andM.-R. Kula, 1994, Bioorg. Med. Chem. 2:421-428; T. Zelinski et al.,1994, J. Biotechnol. 33(3):283-92). Additionally, Baker's yeast has beenwidely used for the asymmetric reductive biotransformation of a varietyof 2-ketones and 3-ketones (R. Csuz and B. I. Glanzer, 1991, Chem. Rev.91:49-97; R. Devaux-Basseguy et al., 1997, Enzyme Microb. Technol.20:248-258; W. Hummel and M.-R. Kula, 1989, Eur. J. Biochem. 184:1-13;W. Kruse et al., 1996, Recl. Trav. Chim. Pays-Bas 115:239-243; T. Lovinyet al., 1985, Biochem. J. 230:579-85; W. F. H. Sybesma et al., 1998,Biocatal. Biotransform. 16:95-134; O. P. Ward and C. S. Young, 1990,Enzyme Microb. Technol. 12:482-493).

Similarly, Gluconobacter oxydans cells have been used in the reductionof various ketones to (S)-alcohols with high enantiomeric excess (P.Adlercreutz, 1991, Enzyme Microb. Technol. 13:9-14; P. Adlercreutz,1991, Biotechnol. Lett. 13:229-234). In addition, G. oxydans2-ketoreductase has been purified, and the purified polypeptide has beenpartly sequenced (V. Nanduri et al., 2000, J. Indust. Microbiol.Biotechnol. 25:171-175). However, large-scale synthesis ofS-(+)-2-pentanol requires a large cell mass, i.e., a ratio of2-pentanone to cell mass of 1 kg:50 kg. In accordance with the presentinvention, G. oxydans 2-ketoreductase was purified and cloned foroverexpression in Escherichia coli. The disclosed 2-ketoreductaseexpression system thereby allows industrial production ofS-(+)-2-pentanol and other chiral alcohols.

SUMMARY OF THE INVENTION

It is an object of the present invention to provide a novel2-ketoreductase isolated from the bacterium, Gluconobacter oxydans, andvariants, modifications, and fragments thereof.

It is also an object of the invention to provide isolated G. oxydans2-ketoreductase polynucleotides, e.g., DNA and RNA molecules, comprisingnucleotide sequences encoding G. oxydans 2-ketoreductase polypeptides,as well as nucleic acid variants, modifications, fragments, andcomplementary sequences thereof.

It is a further object of the present invention to provide nucleic acidprobes and primers, as well as vectors and host cells, comprising G.oxydans 2-ketoreductase polynucleotides.

It is yet a further object of the present invention to provide isolated,recombinant G. oxydans 2-ketoreductase, and enzyme fragments, variants,and modifications thereof.

It is another object of the present invention to provide antibodies andantibody fragments that specifically bind to the G. oxydans2-ketoreductase, or enzyme variants, modifications, or fragmentsthereof..

It is yet another object of the present invention to provide methods ofusing the G. oxydans 2-ketoreductase polynucleotides, vectors, and hostcells to produce G. oxydans 2-ketoreductase.

It is still another object of the present invention to provide methodsof using the recombinant G. oxydans 2-ketoreductase in enzymaticreactions requiring the synthesis of chiral alcohols. In variousaspects, this process uses cell-free extracts or whole cells expressingrecombinant G. oxydans 2-ketoreductase.

It is a further object of the present invention to provide methods ofpurifying the G. oxydans 2-ketoreductase, or enzyme variants,modifications, or fragments thereof, using the disclosed antibodies orantibody fragments.

Additional objects and advantages afforded by the present invention willbe apparent from the detailed description and exemplificationhereinbelow.

DESCRIPTION OF THE FIGURES

The appended drawings of the figures are presented to further describethe invention and to assist in its understanding through clarificationof its various aspects. In the figures of the present invention, thenucleotide and amino acid sequences are represented by their one-letterabbreviations.

FIGS. 1-1 to 1-3 the nucleotide and encoded amino acid sequence of theGluconobacter oxydans 2-ketoreductase gene. The bottom line shows thenucleotide sequence (SEQ ID NO:1); the top line shows the amino acidsequence (SEQ ID NO:2). In the nucleotide sequence, “Y”=C+T; “R”=A+G;“I”=deoxyinosine; “M”=A+C; “V”=A+C+G; “B”=C+T+G; “S”=C+G; “D”=A+T+G;“K”=T+G; and “N”=A+T+C+G.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to nucleotide sequences that comprise thenon-coding and protein-coding regions for a Gluconobacter oxydans enzymewith ketoreductase activity. The present invention also relates to aminoacid sequences encoded by these protein-coding regions. Also related areisolated nucleic acids and polypeptides comprising the disclosedsequences, as well as reagents (e.g., probes, primers, and antibodies)relating to these sequences. The G. oxydans nucleic acids andpolypeptides of the present invention are useful for variousbiotechnology and pharmaceutical applications as disclosed in detailherein.

DEFINITIONS

Use of the terms “SEQ ID NO:9-SEQ ID NO:10” etc., is intended, forconvenience, to refer to each individual SEQ ID NO. individually, and isnot intended to refer to the sequences collectively. The inventionencompasses each sequence individually, as well as any combinationthereof.

“Nucleic acid or “polynucleotide” as used herein refers to purine- andpyrimidine-containing polymers of any length, either polyribonucleotidesor polydeoxyribonucleotide or mixed polyribo-polydeoxyribonucleotides.This includes single-and double-stranded molecules, i.e., DNA-DNA,DNA-RNA and RNA-RNA hybrids, as well as “protein nucleic acids” (PNA)formed by conjugating bases to an amino acid backbone. This alsoincludes nucleic acids containing modified bases. Polynucleotides, e.g.,oligonucleotides, include naturally-occurring species or syntheticspecies formed from naturally-occurring subunits or their closehomologs. The term may also refer to moieties that function similarly topolynucleotides, but have non-naturally-occurring portions. Thus,polynucleotides may have altered sugar moieties or inter-sugar linkages.Exemplary among these are phosphorothioate and other sulfur containingspecies which are known in the art.

A “coding sequence” or a “protein-coding sequence” is a polynucleotidesequence capable of being transcribed into mRNA and/or capable of beingtranslated into a polypeptide. The boundaries of the coding sequence aretypically determined by a translation start codon at the 5′-terminus anda translation stop codon at the 3′-terminus.

A “complement” or “complementary sequence” of a nucleic acid sequence asused herein refers to the antisense sequence that participates inWatson-Crick base-pairing with the original sequence.

A “probe” or “primer” refers to a nucleic acid or oligonucleotide thatforms a hybrid structure with a sequence in a target region due tocomplementarily of at least one sequence in the probe or primer with asequence in the target region.

“Isolated”, as used herein, refers to a substantially purified G.oxydans molecule (e.g., nucleic acid, polypeptide, peptide, proteinfusion, or antibody) that is substantially free of cellular material,culture medium, or other components. Such isolated molecules containless than 50%, preferably less than 25%, more preferably less than 10%,and most preferably less than 1% of the components with which they wereassociated.

The term “vector” as used herein refers to a nucleic acid moleculecapable of replicating itself and another nucleic acid molecule to whichit has been linked. A vector, for example, can be a plasmid, recombinantvirus, or transposon.

“Host” includes prokaryotes and eukaryotes. The term includes anorganism or cell that is the recipient of a replicable vector.

A “recombinant” G. oxydans polypeptide or peptide refers to an aminoacid sequence encoded by a G. oxydans nucleotide sequence describedherein.

As used herein, the terms “protein” and “polypeptide” are synonymous.“Peptides” are defined as fragments or portions of polypeptides,preferably fragments or portions having at least one functional activity(e.g., catalytic or antigenic activity) as the complete polypeptidesequence.

The term “antigenic” refers to the ability of a molecule (e.g., apolypeptide or peptide) to bind to its specific antibody, or an antibodyfragment, with sufficiently high affinity to form a detectableantigen-antibody complex.

A “sample” as used herein refers to a biological sample, for example,cells, cell culture media, cell components (e.g., cell membranes orcellular organelles), cell extracts (e.g., cytoplasm, cytosol, ornuclear extracts), as well as samples obtained from, for-example, alaboratory procedure.

General descriptions of the foregoing terms and others are known in theart. See, e.g., Roitt et al., 1989, Immunology, 2^(nd) Edition, C. V.Mosby Company, New York; Male et al., 1991, Advanced Immunology, 2^(nd)Edition, Grower Medical Publishing, New York.

Nucleic Acids

One aspect of the present invention pertains to isolated G. oxydans2-ketoreductase nucleic acids having a nucleotide sequence such as SEQID NO:1, or variants, modifications, fragments, or complementarysequences thereof. The nucleic acid molecules of the invention can beDNA or RNA (e.g., DNA, RNA, DNA/DNA, and DNA/RNA). A preferred nucleicacid is a DNA encoding a G. oxydans 2-ketoreductase (SEQ ID NO:2), orfragments or functional equivalents thereof. Such nucleic acids cancomprise at least 15, 20, 21, 25, 50, 100, 200, 250, 300, 400, 500, or1000 contiguous nucleotides.

The term “isolated” as used throughout this application refers to a G.oxydans 2-ketoreductase nucleic acid, polypeptide, peptide, proteinfusion, or antibody, that is substantially free of cellular material,culture medium, or other components. An isolated or substantiallypurified molecule contains less than about 50%, preferably less thanabout 25%, more preferably less than about 10%, and most preferably lessthan 1% of the components with which it was associated.

The term “functional equivalent” is intended to include nucleotidesequences encoding functionally equivalent G. oxydans 2-ketoreductase. Afunctional equivalent of a G. oxydans 2-ketoreductase includes fragmentsor variants that perform at least one characteristic function of theenzyme (e.g., catalysis or antigenicity). For example, DNA sequencepolymorphisms within the nucleotide sequence of a G. oxydans2-ketoreductase polypeptide, especially those within the third base of acodon, may result in “silent” mutations, which do not affect the encodedamino acid sequence of the polypeptide due to the degeneracy of thegenetic code.

Preferred embodiments include an isolated nucleic acid sharing at least50, 54, 55, 60, 70, 77, 80, 85, 90, 95, 99, or 100% sequence identitywith a polynucleotide sequence of G. oxydans 2-ketoreductase (e.g., SEQID NO:1). This polynucleotide sequence may be identical to thenucleotide sequence of G. oxydans 2-ketoreductase (e.g., SEQ ID NO:1),or may include up to a certain integer number of nucleotide alterationsas compared to the reference sequence.

“Identity,” as known in the art, is a relationship between two or morepolypeptide sequences or two or more polynucleotide sequences, asdetermined by comparing the sequences. In the art, “identity” also meansthe degree of sequence relatedness between polypeptide or polynucleotidesequences, as the case may be, as determined by the match betweenstrings of such sequences. “Identity” and “similarity” can be readilycalculated by known methods, including but not limited to thosedescribed in Lesk, A. M. (Ed.), 1988, Computational Molecular Biology,Oxford University Press, New York; Smith, D. W. (Ed.), 1993,Biocomputing. Informatics and Genome Projects, Academic Press, New York;Griffin, A. M., and Griffin, H. G. (Eds.), 1994, Computer Analysis ofSequence Data, Part I, Humana Press, New Jersey; von Heinje, G., 1987,Sequence Analysis in Molecular Biology, Academic Press; Gribskov, M. andDevereux, J. (Eds.), 1991, Sequence Analysis Primer, M. Stockton Press,New York; and Carillo, H., and Lipman, D., 1988, SIAM J. Applied Math.48:1073.

For nucleic acids, sequence identity can be determined by comparing aquery sequences to sequences in publicly available sequence databases(NCBI) using the BLASTN2 algorithm (S. F. Altschul et al., 1997, Nucl.Acids Res., 25:3389-3402). The parameters for a typical search are:E=0.05, v=50, B=50, wherein E is the expected probability score cutoff,V is the number of database entries returned in the reporting of theresults, and B is the number of sequence alignments returned in thereporting of the results (S. F. Altschul et al., 1990, J. Mol. Biol.,215:403-410).

In another approach, nucleotide sequence identity can be calculatedusing the following equation: % identity=(number of identicalnucleotides)/(alignment length in nucleotides)*100. For thiscalculation, alignment length includes internal gaps but not includesterminal gaps. Alternatively, nucleotide sequence identity can bedetermined experimentally using the specific hybridization conditionsdescribed below.

In accordance with the present invention, nucleic acid alterations areselected from the group consisting of at least one nucleotide deletion,substitution, including transition and transversion, insertion, ormodification (e.g., via RNA or DNA analogs, dephosphorylation,methylation, or labeling). Alterations may occur at the 5′ or 3′terminal positions of the reference nucleotide sequence or anywherebetween those terminal positions, interspersed either individually amongthe nucleotides in the reference sequence or in one or more contiguousgroups within the reference sequence. Alterations of a nucleic acidsequence of G. oxydans 2-ketoreductase (e.g., SEQ ID NO:1) may createnonsense, missense, or frameshift mutations in the coding sequence, andthereby alter the polypeptide encoded by the nucleic acid.

The present invention also encompasses naturally-occurring nucleotidepolymorphisms of G. oxydans 2-ketoreductase (e.g., SEQ ID NO:1). As willbe understood by those in the art, the genomes of all organisms undergospontaneous mutation in the course of their continuing evolutiongenerating variant forms of gene sequences (Gusella, 1986, Ann. Rev.Biochem. 55:831-854). Restriction fragment length polymorphisms (RFLPs)include variations in DNA sequences that alter the length of arestriction fragment in the sequence (Botstein et al., 1980, Am. J. Hum.Genet 32, 314-331). Short tandem repeats (STRs) include tandem di-, tri-and tetranucleotide repeated motifs, also termed variable number tandemrepeat (VNTR) polymorphisms.

Single nucleotide polymorphisms (SNPs) are far more frequent than RFLPS,STRs, and VNTRs. SNPs may occur in protein coding (e.g., exon), ornon-coding (e.g., intron, 5′UTR, and 3′UTR) sequences. SNPs in proteincoding regions may comprise silent mutations that do not alter the aminoacid sequence of a protein. Alternatively, SNPs in protein codingregions may produce conservative or non-conservative amino acid changes,described in detail below. In non-coding sequences, SNPs may also resultin defective protein expression (e.g., as a result of defectivesplicing). Other single nucleotide polymorphisms have no phenotypiceffects.

Further encompassed by the present invention are nucleic acid moleculesthat share moderate homology with the G. oxydans 2-ketoreductase nucleicacid sequence (e.g., SEQ ID NO:1 or a complementary sequence), andhybridize to a G. oxydans 2-ketoreductase nucleic acid molecule undermoderate stringency hybridization conditions. More preferred are nucleicacid molecules that share substantial homology with a G. oxydans2-ketoreductase nucleic acid sequence (e.g., SEQ ID NO:1 or acomplementary sequence) and hybridize to G. oxydans 2-ketoreductasenucleic acid molecules under high stringency hybridization conditions.

As used herein, the phrase “moderate homology” refers to sequences whichshare at least 60% sequence identity with a ketoreductase sequence(e.g., SEQ ID NO:1), whereas the phrase “substantial homology.” refersto sequences that share at least 90% sequence identity with aketoreductase sequence. It is recognized, however, that polypeptides andthe nucleic acids encoding such polypeptides containing less than theabove-described level of homology arising as splice variants or that aremodified by conservative amino acid substitutions (or substitution ofdegenerate codons) are contemplated to be within the scope of thepresent invention.

The phrase “hybridization conditions” is used herein to refer toconditions under which a double-stranded nucleic acid hybrid is formedfrom two single nucleic acid strands, and remains stable. As known tothose of skill in the art, the stability of the hybrid sequence isreflected in the melting temperature (T_(m)) of the hybrid (see F. M.Ausubel et al. (Eds.), 1995, Current Protocols in Molecular Biology,John Wiley and Sons, Inc., New York, N.Y.). The T_(m) decreasesapproximately 0.5° C. to 1.5° C. with every 1% decrease in sequencehomology. In general, the stability of a hybrid sequence is a functionof the length and guanine/cytosine content of the hybrid, the sodium ionconcentration, and the incubation temperature. Typically, thehybridization reaction is initially performed under conditions of lowstringency, followed by washes of varying, but higher, stringency.Reference to hybridization stringency relates to such washingconditions.

In accordance with the present invention, “high stringency” conditionscan be provided, for example, by hybridization in 50% formamide, 5×Denhardt's solution, 5×SSPE, and 0.2% SDS at 42° C., followed by washingin 0.1×SSPE and 0.1% SDS at 65° C. By comparison, “moderate stringency”can be provided, for example, by hybridization in 50% formamide, 5×Denhardt's solution, 5×SSPE, and 0.2% SDS at 42° C., followed by washingin 0.2×SSPE and 0.2% SDS at 65° C. In addition, “low stringency”conditions can be provided, for example, by hybridization in 10%formamide, 5× Denhardt's solution, 6×SSPE, and 0.2% SDS at 42° C.,followed by washing in 1×SSPE and 0.2% SDS at 50° C. It is understoodthat these conditions may be varied using a variety of buffers andtemperatures well known to those skilled in the art.

In a preferred embodiment of the present invention, the nucleic acid isa DNA molecule encoding at least a fragment of a G. oxydans2-ketoreductase (SEQ ID NO:2). A nucleic acid molecule encoding a G.oxydans 2-ketoreductase can be obtained from mRNA present inGluconobacter oxydans cells. It may also be possible to obtain nucleicacid molecules encoding a 2-ketoreductase from Gluconobacter oxydansgenomic DNA. In addition, a nucleic acid encoding a G. oxydans2-ketoreductase can be cloned from either a cDNA or a genomic library inaccordance with the protocols described in detail herein.

Nucleic acids encoding G. oxydans 2-ketoreductase enzymes can also becloned using established polymerase chain reaction (PCR) techniques (seeK. Mullis et al., 1986, Cold Spring Harbor Symp. Quant. Biol. 51:260; K.H. Roux, 1995, PCR Methods Appl. 4:S185) in accordance with the nucleicacid sequence information provided herein. For example, PCR techniquescan be used to produce the nucleic acids of the invention, using eitherRNA (e.g., mRNA) or DNA (e.g., genomic DNA) as templates. Primers usedfor PCR can be synthesized using the sequence information providedherein and can further be designed to introduce appropriate newrestriction sites, if desirable, to facilitate incorporation into agiven vector for recombinant expression.

The nucleic acid molecules of the invention, or fragments thereof, canalso be chemically synthesized using standard techniques. Variousmethods of chemically synthesizing polydeoxynucleotides are known,including solid-phase synthesis which, like peptide synthesis, has beenfully automated in commercially available DNA synthesizers (see, forexample, U.S. Pat. No. 4,598,049 to Itakura et al.; U.S. Pat. No.4,458,066 to Caruthers et al.; U.S. Pat. Nos. 4,401,796 and 4,373,071 toItakura).

It will be appreciated by one skilled in the art that variations in oneor more nucleotides (up to about 3-4% of the nucleotides) of the nucleicacid molecules encoding a G. oxydans 2-ketoreductase may exist amongorganisms within a population due to natural allelic variation. Any -andall such nucleotide variations and resulting amino acid polymorphismsare within the scope of the invention. Furthermore, there may be one ormore isoforms or related family members of the G. oxydans2-ketoreductase described herein. Such isoforms or family members aredefined as polypeptides that are related in function and amino acidsequence to a G. oxydans 2-ketoreductase (e.g., SEQ ID NO:2), butencoded by genes at different loci. In addition, it is possible tomodify the DNA sequence of the G. oxydans 2-ketoreductase gene usinggenetic techniques to produce proteins or peptides with altered aminoacid sequences.

DNA sequence mutations can be introduced into a nucleic acid encoding aG. oxydans 2-ketoreductase by any one of a number of methods, includingthose for producing simple deletions or insertions, systematicdeletions, insertions or substitutions of clusters of bases orsubstitutions of single bases, to generate desired variants. Mutationsof the G. oxydans 2-ketoreductase nucleic acid molecule to generateamino acid substitutions or deletions are preferably obtained bysite-directed mutagenesis.

Site directed mutagenesis systems are well known in the art, and can beobtained from commercial sources (see, for example, Amersham-PharmaciaBiotech, Inc., Piscataway, N.J.). Guidance in determining which aminoacid residues may be substituted, inserted, or deleted withoutabolishing biological or immunological activity may be found usingcomputer programs well known in the art, for example, DNASTAR software(DNASTAR, Inc., Madison, Wis.). Mutant forms of the G. oxydans2-ketoreductase nucleic acid molecules are considered within the scopeof the present invention, where the expressed polypeptide or peptide iscapable catalytic or antigenic activity.

A fragment of the nucleic acid molecule encoding a G. oxydans2-ketoreductase is defined as a nucleotide sequence having fewernucleotides than the nucleotide sequence encoding the entire amino acidsequence of the enzyme. In one embodiment of the present invention, anucleic acid molecule corresponding to a fragment of a G. oxydans2-ketoreductase nucleic acid sequence can be used as a probe forassaying a biological sample (e.g., from cells or cell extracts), theexpression of one or more enzymes, or as a primer for DNA sequencing orPCR amplification. Preferably, such fragments are at least 8, 12, 15,20, 21, or 25 contiguous nucleotides in length.

In certain embodiments, the nucleic acid molecules of the invention mayinclude linker sequences, modified restriction endonuclease sites, andother sequences useful for molecular cloning, expression, orpurification of recombinant protein or fragments thereof. Nucleic acidmolecules in accordance with the present invention may also beconjugated with radioisotopes, or chemiluminescent, fluorescent, orother labeling compounds (e.g., digoxigenin). In addition, the nucleicacid molecules of the present invention may be modified by nucleic acidmodifying enzymes, for example, kinases or phosphatases. These and othermodifications of nucleic acid molecules are well known in the art. Inaddition, a nucleic acid molecule that encodes a G. oxydans2-ketoreductase, or a functional fragment thereof, can be ligated to aheterologous sequence to encode a fusion protein (also called a chimericprotein) as described in detail herein.

Vectors and Host Cells

Another aspect of the present invention pertains to vectors comprising anucleic acid encoding a G. oxydans 2-ketoreductase, as described herein,operably linked to at least one regulatory sequence. “Operably linked”is intended to mean that the nucleotide acid sequence is linked to aregulatory sequence in a manner that allows expression of the nucleotidesequence (i.e., production of mRNA and/or amino acid sequences).Regulatory sequences are known in the art and are selected to directexpression of the desired protein in an appropriate host cell orcell-free expression system. Accordingly, the term regulatory sequenceincludes promoters, enhancers and other expression control elements (seeD. V. Goeddel, 1990, Methods Enzymol. 185:3-7). It should be understoodthat the design of the expression vector may depend on such factors asthe choice of the host cell or expression system to be utilized and/orthe type of polypeptide desired to be expressed.

Suitable expression vectors include, but are not limited to, pUC,pBluescript (Stratagene), pET (Novagen, Inc., Madison, Wis.), as well aspREP, pSE420, and pLEX (Invitrogen). Vectors can contain one or morereplication and inheritance systems for cloning or expression, one ormore markers for selection in the host, e.g. antibiotic resistance, andone or more expression cassettes. The inserted coding sequences can besynthesized by standard methods, isolated from natural sources, orprepared as hybrids. Ligation of the coding sequences to transcriptionalregulatory elements (e.g., promoters,.enhancers, and/or insulators)and/or to other amino acid encoding sequences can be carried out usingestablished methods.

Preferred replication and inheritance systems include M13, ColE1, SV40,baculovirus, lambda, adenovirus, CEN ARS, 2 μm, ARS, and the like.Several regulatory elements (e.g., promoters) have been isolated andshown to be effective in the transcription and translation ofheterologous proteins in the various hosts. Such regulatory regions,methods of isolation, manner of manipulation, etc. are known in the art.Non-limiting examples of bacterial promoters include the β-lactamase(penicillinase) promoter; lactose promoter; tryptophan (trp) promoter;araBAD (arabinose) operon promoter; lambda-derived P₁ promoter and Ngene ribosome binding site; and the hybrid tac promoter derived fromsequences of the trp and lac UV5 promoters.

Non-limiting examples of yeast promoters include the 3-phosphoglyceratekinase promoter, glyceraldehyde-3-phosphate dehydrogenase (GAFDH or GAP)promoter, galactokinase (GAL1) promoter, galactoepimerase promoter, andalcohol dehydrogenase (ADH1) promoter. Suitable promoters for mammaliancells include, without limitation, viral promoters, such as those fromSimian Virus 40 (SV40), Rous sarcoma virus (RSV), adenovirus (ADV), andbovine papilloma virus (BPV). Alternatively, the endogenous G. oxydansregulatory elements (e.g., in SEQ ID NO:1) can be used.

Eukaryotic cells may also require terminator sequences, polyadenylationsequences, and enhancer sequences that modulate gene expression.Sequences that cause amplification of the gene may also be desirable.These sequences are well known in the art. Furthermore, sequences thatfacilitate secretion of the recombinant product from cells, including,but not limited to, bacteria, yeast, and animal cells, such as secretorysignal sequences and/or preprotein or proprotein sequences, may also beincluded in accordance with established methods. Secretory signalsequences are generally positioned 5′ to the nucleotide sequenceencoding the protein of interest, although certain signal sequences canbe positioned 3′ to the nucleotide sequence of interest (see, e.g.,Welch et al., U.S. Pat. No. 5,037,743; Holland et al., U.S. Pat. No.5,143,830). Cell-specific secretory signals can be used with certaincell types (e.g., yeast cells).

Expression and cloning vectors will likely contain a selectable marker,a gene encoding a protein necessary for survival or growth of a hostcell transformed with the vector. The presence of this gene ensuresgrowth of only those host cells that express the inserts. Typicalselection genes encode proteins that 1) confer resistance to antibioticsor other toxic substances, e.g., ampicillin, neomycin, methotrexate,etc.; 2) complement auxotrophic deficiencies, or 3) supply criticalnutrients not available from complex media, e.g., the gene encodingD-alanine racemase for Bacilli. Markers may be an inducible ornon-inducible gene and will generally allow for positive selection.Non-limiting examples of markers include the ampicillin resistancemarker (i.e., beta-lactamase), tetracycline resistance marker,neomycin/kanamycin resistance marker (i.e., neomycinphosphotransferase), dihydrofolate reductase, glutamine synthetase, andthe like. The choice of the proper selectable marker will depend on thehost cell, and appropriate markers for different hosts as understood bythose of skill in the art.

Suitable cell-free expression systems for use with the present inventioninclude, without limitation, rabbit reticulocyte lysate, wheat germextract, canine pancreatic microsomal membranes, E. coli S30 extract,and coupled transcription/translation systems (Promega Corp., Madison,Wis.). Suitable host cells include bacteria, fungi, yeast, plant,insect, and animal, mammalian, and human cells. Specifically includedare SF9, C129, 293, NIH 3T3, CHO, COS, HeLa, and Neurospora cells.Insect cell systems (i.e., lepidopteran host cells and baculovirusexpression vectors) (Luckow and Summers, 1988, Biotechnology 6:47-55)are also included.

Preferred host cells include fungal cells, such as Aspergillus (A.niger, A. oryzae, and A. fumigatus), Fusarium venenatum,Schizosaccharomyces pombe, Saccharomyces cerevisiae, Kluyveromyceslactis, Kluyveromyces fragilis, Ustilago maydis, Candida (e.g., C.albicans, C. methylica, C. boidinii, C. tropicalis, C. wickerhamii, C.maltosa, and C. glabrata), Hansenula (e.g., H. anomala, H. polymorpha,H. wingei, H. jadinii and H. satumus,); and Pichia (e.g., P. angusta, P.pastoris, P. anomala, P. stipitis, P. methanolica, and P.guilliermondii) cells. Particularly preferred are bacterial cells, suchas Staphylococcus aureus, Escherichia coli, Bacillus (e.g., B.licheniformis, B. amyloliquefaciens, and B. subtilis) and Streptomyces(e.g., Streptomyces lividans and Streptomyces coelicolor) cells.

In general, host cells can be transformed, transfected, or infected asappropriate by any suitable method including electroporation, calciumchloride-, lithium chloride-, lithium acetate/polyethylene glycol-,calcium phosphate-, DEAE-dextran-, liposome-mediated DNA uptake,spheroplasting, injection, microinjection, microprojectile bombardment,phage infection, viral infection, or other established methods.Alterniatively, vectors containing the nucleic acids of interest can betranscribed in vitro, and the resulting RNA introduced into the hostcell by well-known methods, e.g., by injection (see, Kubo et al., 1988,FEBS Letts. 241:119).

Methods for transforming S. cerevisiae cells with exogenous DNA andproducing recombinant proteins therefrom are found in, for example,Kawasaki, U.S. Pat. No. 4,599,311; Kawasaki et al., U.S. Pat. No.4,931,373; Brake, U.S. Pat. No. 4,870,008; Welch et al., U.S. Pat. No.5,037,743; Murray et al., U.S. Pat. No. 4,845,075, and Kawasaki et al.,U.S. Pat. No. 4,931,373). Transformation methods for other yeasts,including H. polymorpha/P. angusta, S. pombe, K. lactis, K. fragilis, U.maydis, P. pastoris, P. methanolica/C. methylica, and C. maltosa areknown in the art (see, for example, Gleeson et al., 1986, J. Gen.Microbiol. 132:3459-3465; Cregg, U.S. Pat. No. 4,882,279; and Hiep etal., 1993, Yeast 9:1189-1197). Aspergillus cells can be transformedaccording to the methods of McKnight et al., U.S. Pat. No. 4,935,349,while Acremonium chrysogenum cells can be transformed in accordance withSumino et al., U.S. Pat. No. 5,162,228. In general, host cells mayintegrate the nucleic acid molecules of this invention into chromosomalloci. Alternatively, the host cells may maintain the nucleic acidmolecules via episomal vectors.

In one embodiment, an expression vector comprises a nucleic acidencoding at least a fragment of a G. oxydans 2-ketoreductase. In anotherembodiment, the expression vector comprises a DNA sequence encoding atleast a fragment of a G. oxydans 2-ketoreductase fused in-frame to a DNAsequence encoding a heterologous polypeptide or peptide. Such expressionvectors can be used to transfect host cells to thereby produce G.oxydans 2-ketoreductase polypeptides or peptides, including fusionproteins or peptides encoded by nucleic acid molecules as describedbelow.

Several well-established techniques can be used to determine theexpression levels and pattems of G. oxydans 2-ketoreductase. Forexample, mRNA levels can be determined utilizing Northern blot analysis(J. C. Alwine et al., 1977, Proc. Natl. Acad. Sci. USA. 74:5350-5354; I.M. Bird, 1998, Methods Mol. Biol. 105:325-36), whereby poly(A)⁺ RNA isisolated from cells, separated by gel electrophoresis, blotted onto asupport surface (e.g., nitrocellulose or Immobilon-Ny+ (Millipore Corp.,Bedford, Mass.)), and incubated with a labeled (e.g., fluorescentlylabeled or radiolabeled) oligonucleotide probe that is capable ofhybridizing with the mRNA of interest.

Alternatively, mRNA levels can be determined by quantitative (forreview, see W. M. Freeman et al., 1999, Biotechniques 26:112-122) orsemi-quantitative RT-PCR analysis (Ren et al., Mol. Brain Res.59:256-63). In accordance with this technique, poly(A)⁺ RNA is isolatedfrom cells, used for cDNA synthesis, and the resultant cDNA is incubatedwith PCR primers that are capable of hybridizing with the template andamplifying the template sequence to produce levels of the PCR productthat are proportional to the cellular levels of the mRNA of interest.Another technique, in situ hybridization, can also be used to determinemRNA levels (reviewed by A. K. Raap, 1998, Mutat Res. 400:287-298). Insitu hybridization techniques allow the visual detection of mRNA in acell by incubating the cell with a labeled (e.g., fluorescently labeledor digoxigenin labeled) oligonucleotide probe that hybridizes to themRNA of interest, and then examining the cell by microscopy.

G. oxydans 2-ketoreductase fragments, modifications, or variants can bealso be assessed directly by well-established techniques. For example,host cell expression of the recombinant polypeptides can be evaluated bywestern blot analysis using antibodies specifically reactive with thesepolypeptides (see above). Production of secreted forms of thepolypeptides can be evaluated by immunoprecipitation using monoclonalantibodies that are specifically reactive the polypeptides. Other, morepreferred, assays take advantage of the functional characteristics of G.oxydans 2-ketoreductase. As previously set forth, G. oxydans2-ketoreductase can be used in various reactions to generate chiralalcohols. Thus, G. oxydans 2-ketoreductase function can be assessed bymeasuring the products of these reactions. In specific aspects, any oneof the assays described hereinbelow can be employed.

Polypeptides

A further aspect of the present invention pertains to G. oxydans2-ketoreductase polypeptides (e.g., recombinant polypeptides). Thepresent invention encompasses a G. oxydans 2-ketoreductase polypeptide(e.g., SEQ ID NO:2), and fragments and functional equivalents thereof. .Such polypeptides can comprise at least 5, 12, 20, 21, 25, 30, 32, 35,50, 100, 170, 200, 210, 300, or 500 contiguous amino acid residues.Preferred are polypeptides that share moderate homology with a G.oxydans 2-ketoreductase polypeptide (e.g., SEQ ID NO:2). More preferredare polypeptides that share substantial homology with a G. oxydans2-ketoreductase polypeptide.

The term “functional equivalent” is intended to include proteins whichdiffer in amino acid sequence from the G. oxydans 2-ketoreductasepolypeptide (e.g., SEQ ID NO:2), but where such differences result in amodified protein which performs at least one characteristic function ofpolypeptide (e.g., catalytic or antigenic activity). For example, afunctional equivalent of a G. oxydans 2-ketoreductase polypeptide mayhave a modification such as a substitution, addition or deletion of anamino acid residue which is not directly involved in the function ofthis polypeptide. Various modifications of the G. oxydans2-ketoreductase polypeptide to produce functional equivalents of thesepolypeptides can be made in accordance with established methods.

It is also possible to modify the structure of a G. oxydans2-ketoreductase polypeptide for such purposes as increasing solubility,enhancing reactivity, or increasing stability (e.g., shelf life ex vivoand resistance to proteolytic degradation in vivo). Such modifiedproteins are considered functional equivalents of a G. oxydans2-ketoreductase polypeptide as defined herein. Preferably, G. oxydans2-ketoreductase polypeptides are modified so that they retain catalyticactivity. Those residues shown to be essential for activity can bemodified by replacing the essential amino acid with another, preferablysimilar amino acid residue (a conservative substitution) whose presenceis shown to enhance, diminish, but not eliminate, or not effect receptorinteraction. In addition, those amino acid residues that are notessential for catalysis can be modified by being replaced by anotheramino acid whose incorporation may enhance, diminish, or not effectreactivity.

In order to enhance stability and/or reactivity, a G. oxydans2-ketoreductase polypeptide can be altered to incorporate one or morepolymorphisms in the amino acid sequence. Additionally, D-amino acids,non-natural amino acids, or non-amino acid analogs can be substituted oradded to produce a modified polypeptide. Furthermore, the polypeptidesdisclosed herein can be modified using polyethylene glycol (PEG)according to known methods (S. I. Wie et al., 1981, Int. Arch. AllergyAppl. Immunol. 64(1):84-99) to produce a protein conjugated with PEG. Inaddition, PEG can be added during chemical synthesis of the protein.Other possible modifications include phosphorylation, sulfation,reduction/alkylation (Tarr, 1986, Methods of ProteinMicrocharacterization, J. E. Silver (Ed.) Humana Press, Clifton, N.J.,pp. 155-194); acylation (Tarr, supra); chemical coupling (Mishell andShiigi (Eds.), 1980, Selected Methods in Cellular Immunology, W HFreeman, San Francisco, Calif.; U.S. Pat. No. 4,939,239); and mildformalin treatment (Marsh, 1971, Int. Arch. of Allergy and Appl.Immunol. 41:199-215).

Modified polypeptides can have conservative changes, wherein asubstituted amino acid has similar structural or chemical properties,e.g., replacement of leucine with isoleucine. More infrequently, amodified polypeptide can have non-conservative changes, e.g.,substitution of a glycine with a tryptophan. Guidance in determiningwhich amino acid residues can be substituted, inserted, or deletedwithout abolishing biological or immunological activity can be foundusing computer programs well known in the art, for example, DNASTARsoftware (DNASTAR, Inc., Madison, Wis.)

As non-limiting examples, conservative substitutions in the G. oxydans2-ketoreductase amino acid sequence can be made in accordance with thefollowing table: Original Residue Conservative Substitution(s) Ala SerArg Lys Asn Gln, His Asp Glu Cys Ser Gln Asn Glu Asp Gly Pro His Asn,Gln Ile Leu, Val Leu Ile, Val Lys Arg, Gln, Glu Met Leu, Ile Phe Met,Leu, Tyr Ser Thr Thr Ser Trp Tyr Tyr Trp, Phe Val Ile, Leu

Substantial changes in function or immunogenicity can be made byselecting substitutions that are less conservative than those shown inthe table, above. For example, non-conservative substitutions can bemade which more significantly affect the structure of the polypeptide inthe area of the alteration, for example, the alpha-helical, orbeta-sheet structure; the charge or hydrophobicity of the molecule atthe target site; or the bulk of the side chain. The substitutions whichgenerally are expected to produce the greatest changes in thepolypeptide's properties are those where 1) a hydrophilic residue, e.g.,seryl or threonyl, is substituted for (or by) a hydrophobic residue,e.g., leucyl, isoleucyl, phenylalanyl, valyl, or alanyl; 2) a cysteineor proline is substituted for (or by) any other residue; 3) a-residuehaving an electropositive side chain, e.g., lysyl, arginyl, or histidyl,is substituted for (or by) an electronegative residue, e.g., glutamyl oraspartyl; or 4) a residue having a bulky side chain, e.g.,phenylalanine, is substituted for (or by) a residue that does not have aside chain, e.g., glycine.

Preferred polypeptide embodiments further include an isolatedpolypeptide comprising an amino acid sequence sharing at least 50, 54,55, 60, 70, 80, 85, 86, 90, 95, 97, 98, 99, 99.5 or 100% identity withthe amino acid sequence of G. oxydans 2-ketoreductase (SEQ ID NO:2).This polypeptide sequence may be identical to the sequence of G. oxydans2-ketoreductase (SEQ ID NO:2), or may include up to a certain integernumber of amino acid alterations as compared to the reference sequence

Percent sequence identity can be calculated using computer programs ordirect sequence comparison. Preferred computer program methods todetermine identity between two sequences include, but are not limitedto, the GCG program package, FASTA, BLASTP, and TBLASTN (see, e.g., D.W. Mount, 2001, Bioinformatics: Sequence and Genome Analysis,

Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.). TheBLASTP and TBLASTN programs are publicly available from NCBI and othersources. The well-known Smith Waterman algorithm may also be used todetermine identity.

Exemplary parameters for amino acid sequence. comparison include thefollowing: 1) algorithm from Needleman and Wunsch, 1970, J Mol. BioL48:443-453; 2) BLOSSUM62 comparison matrix from Hentikoff and Hentikoff,1992, Proc. Natl. Acad. Sci. USA 89:10915-10919; 3) gap penalty=12; and4) gap length penalty=4. A program useful with these parameters ispublicly available as the “gap” program (Genetics Computer Group,Madison, Wis.). The aforementioned parameters are the default parametersfor polypeptide comparisons (with no penalty for end gaps).Altematively, polypeptide sequence identity can be calculated using thefollowing equation: % identity=(the number of identicalresidues)/(alignment length in amino acid residues)*100. For thiscalculation, alignment length includes internal gaps but does notinclude terminal gaps.

In accordance with the present invention, polypeptide sequences may beidentical to the sequence of G. oxydans 2-ketoreductase (e.g., SEQ IDNO:2), or may include up to a certain integer number of amino acidalterations. Polypeptide alterations are selected from the groupconsisting of at least one amino acid deletion, substitution, includingconservative and non-conservative substitution, or insertion.Alterations may occur at the amino- or carboxy-terminal positions of thereference polypeptide sequence or anywhere between those terminalpositions, interspersed either individually among the amino acids in thereference sequence or in one or more contiguous groups within thereference sequence. In specific embodiments, polypeptide variants may beencoded by G. oxydans 2-ketoreductase nucleic acids comprising singlenucleotide polymorphisms and/or alternate splice variants.

G. oxydans 2-ketoreductase polypeptides may also be modified byconjugation with a label capable of providing, a detectable signal,either directly or indirectly, including, for example, radioisotopes andfluorescent compounds. Non-limiting examples of fluorescent compoundsinclude Cy3, Cy5, GFP (e.g., EGFP, DsRed, dEFP, etc. (CLONTECH, PaloAlto, Calif.)), Alexa, BODIPY, fluorescein (e.g., FluorX, DTAF, andFITC), rhodamine (e.g., TRITC), auramine, Texas Red, AMCA blue, andLucifer Yellow. Suitable isotopes include, but are not limited to, ³H,¹⁴C, ³²P, ³⁵S, ³⁶Cl, ⁵¹Cr, ⁵⁷Co, ⁵⁸Co, ⁵⁹Fe, ⁹⁰Y, ¹²⁵I, ¹³¹I and ¹⁸⁶Re.

The invention also relates to isolated, synthesized and/or recombinantportions or fragments of a G. oxydans 2-ketoreductase polypeptide (e.g.,SEQ ID NO:2), as described herein. Polypeptide fragments (i.e.,peptides) can be made which have full or partial function on their own,or which when mixed together (though fully, partially, or nonfunctionalalone), spontaneously assemble with one or more other polypeptides toreconstitute a functional protein having at least one functionalcharacteristic of a G. oxydans 2-ketoreductase of this invention. Inaddition, G. oxydans 2-ketoreductase polypeptide fragments may comprise,for example, one or more domains of the polypeptide (e.g., a short chaindehydrogenase domain) disclosed herein.

The polypeptides of the present invention, includingfunction-conservative variants, may be isolated from wild-type or mutantG. oxydans cells, from heterologous organisms or cells (e.g., bacteria,yeast, insect, plant, or mammalian cells) comprising recombinant G.oxydans 2-ketoreductase, or from cell-free translation systems (e.g.,wheat, germ, microsomal membrane, or bacterial extracts) in which a G.oxydans 2-ketoreductase protein-coding sequence has been introduced andexpressed. Furthermore, the polypeptides may be part of recombinantfusion proteins. The polypeptides can also, advantageously, be made bysynthetic chemistry. Polypeptides may be chemically synthesized bycommercially available automated procedures, including, withoutlimitation, exclusive solid phase synthesis, partial solid phasemethods, fragment condensation or classical solution synthesis.

Isolation of Polypeptides

Yet another aspect of the present invention pertains to methods ofisolating G. oxydans 2-ketoreductase polypeptides, or variants,modifications, or fragments thereof from biological samples (e.g.,cells, cell extracts or lysates, cell membranes, growth media, etc.).Fragments of ketoreductase polypeptides (i.e., peptides) includefragments, preferably, having the same or equivalent function oractivity as the full-length polypeptide. Both naturally occurring andrecombinant forms of the G. oxydans 2-ketoreductase polypeptides orpeptides may be used in the methods according to the present invention.Methods for directly isolating and purifying polypeptides or peptidesfrom cellular or extracellular lysates are well known in the art (see E.L. V. Harris and S. Angal (Eds.), 1989, Protein. Purification Methods: APractical Approach, IRL Press, Oxford, England). Such methods include,without limitation, preparative disc-gel electrophoresis, isoelectricfocusing, high-performance liquid chromatography (HPLC), reversed-phaseHPLC, gel filtration, ion exchange and partition chromatography, andcountercurrent distribution, and combinations thereof.

In addition, antibody-based methods can be used to isolate natural orrecombinantly produced G. oxydans 2-ketoreductase polypeptides orpeptides. Antibodies that recognize these polypeptides, or peptidesderived therefrom, can be produced and isolated using methods known andpracticed in the art (see below). G. oxydans 2-ketoreductasepolypeptides or peptides can then be purified from a crude lysate bychromatography on antibody-conjugated solid-phase matrices (see E.Harlow and D. Lane, 1999, Using Antibodies: A Laboratory Manual, ColdSpring Harbor Laboratory, Cold Spring Harbor, N.Y.). Other isolationmethods known and used in the art may also be employed.

To produce recombinant G. oxydans 2-ketoreductase polypeptides orpeptides, DNA sequences encoding the polypeptides or peptides can becloned into a suitable vector for expression in intact host cells or incell-free translation systems as described above (see also J. Sambrooket al., 1989, Molecular Cloning: A Laboratory Manual, 2nd edition, ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y.). DNA sequencescan be optimized, if desired, for more efficient expression in a givenhost organism. For example, codons can be altered to conform to thepreferred codon usage in a given host cell or cell-free translationsystem using techniques routinely practiced in the art.

For some purposes, it may be preferable to produce G. oxydans2-ketoreductase peptides or polypeptides in a recombinant system whereinthe peptides or polypeptides carry additional sequence tags tofacilitate purification. Such markers include epitope tags and proteintags. Non-limiting examples of epitope tags include c-myc,haemagglutinin (HA), polyhistidine (6×-HIS), GLU-GLU, and DYKDDDDK(FLAG®; SEQ ID NO:3) epitope tags. Non-limiting examples of protein tagsinclude glutathione-S-transferase (GST), green fluorescent protein(GFP), and maltose binding protein (MBP).

Epitope and protein tags can be added to peptides by a number ofestablished methods. For example, DNA sequences encoding epitope tagscan be inserted into protein-coding sequences as oligonucleotides or asprimers used in PCR amplification. As an altemative, protein-codingsequences can be cloned into specific vectors that create fusions withepitope tags; for example, pRSET vectors (Invitrogen Corp., San Diego,Calif.). Similarly, protein tags can be added by cloning the codingsequence of a polypeptide or peptide into a vector that creates a fusionbetween the polypeptide or peptide and a protein tag of interest.Suitable vectors include, without limitation, the exemplary plasmids,pGEX (Amersham-Pharmacia Biotech, . Inc., Piscataway, N.J.), pEGFP(CLONTECH Laboratories, Inc., Palo Alto, Calif.), and PMALTM (NewEngland BioLabs, Inc., Beverly, Mass.). Following expression, theepitope or protein tagged polypeptide or peptide can be purified from acrude lysate of the translation system or host cell by chromatography onan appropriate solid-phase matrix. In some cases, it may be preferableto remove the epitope or protein tag (i.e., via protease cleavage)following purification.

In various embodiments, the recombinant G. oxydans 2-ketoreductasepolypeptides are secreted to the cell surface, retained in the cytoplasmof the host cells, or secreted into the growth media. In each case, theproduction of G. oxydans 2-ketoreductase polypeptides can be establishedusing anti-ketoreductase antibodies, or catalytic assays. Thecell-surface and cytoplasmic recombinant G. oxydans 2-ketoreductasepolypeptides can be isolated following cell lysis and extraction ofcellular proteins, while the secreted recombinant G. oxydans2-ketoreductase polypeptides can be isolated from the cell growth mediaby standard techniques (see I. M. Rosenberg (Ed.) 1996, Protein Analysisand Purification: Benchtop Techniques, Birkhauser, Boston, Cam bridge,Mass.).

Methods to improve polypeptide production may include 1) the use ofbacterial expressed fusion proteins comprising signal peptides ortargeting sequences to promote secretion (Tessier et al., 1991, Gene98:177-83; Gamier et al., 1995, Biotechnology 13:1101-4); 2) the use ofserum-free and protein-free culture systems for economical polypeptideproduction (Zang et al., 1995, Biotechnology 13:389-92); 3) the use ofthe eukaryotic regulated secretory pathway for increased production andharvesting efficiency (see Chen et al., 1995, Biotechnology 13:1191-97).Polypeptide production may also be optimized by the utilization of aspecific vector, host cell, expression system, or production protocol,as described in detail herein.

Large-scale microbial protein production can be achieved usingwell-established methods (see, e.g., W. Crueger and A. Crueger, 1990,Biotechnology: A Textbook of Industrial Microbiology Sinauer Associates,Sunderland, Mass. ; A. N. Glazer and H. Nikaido, 1995, Microbialbiotechnology: fundamentals of applied microbiology Freeman, New York,N.Y.; C. M. Brown et al., 1987, Introduction to Biotechnology: BasicMicrobiology, Vol. 10, Blackwell, Oxford, UK). Methods for scaling-upbaculovirus protein production can be found, for example, in R. L. Tomet al., 1995, Methods Mol. Biol. 39:203-24; R. L. Tom et al., 1995,Appl. Microbibl. Biotechnol. 44:53-8; S. A. Weiss, et al., 1995, MethodsMol. Biol. 39:79-95; and C. D. Richardson (Ed.) 1995, BaculovirusExpression Protocols: Methods in Molecular Biology, Vol. 39, HumanaPress, Totowa, N.J. In additional, large-scale protein productionservices are commercially available from, e.g., PanVera Corp., Madison,Wis.; Oxford Expression Technologies, Oxford UK; BioXpress Laboratory,Athens, Ga.; and Recombinant Protein Expression Laboratory, Gainesville,Fla.

In general, large-scale microbial enzyme production systems employ thefollowing procedures. Screens are used to test enzyme activity, pHoptimum, temperature optimum, secretion (downstream processing), and theability to grow the organism in inexpensive large-scale fermentationsystems (high population densities from inexpensive carbon and nitrogenfeedstocks, e.g., corn syrup, molasses, soybean meal, gluten, etc.).Strain improvements are created by random mutagenesis and screening ordirected genetic manipulation (e.g., in Bacillus, Streptomyces,Aspergillus and Saccharomyces strains). For example, mutant strains canprovide 1) relief of repression (e.g., catabolite repression); 2)increased promoter strength; 3) higher affinity ribosome-binding sites;4) higher efficiency of mRNA leader translation; 5) increased mRNA halflife; 6) increased translation efficiency through altered codon usage;7) improvement of secretion efficiency; and 8) increased gene dosage(i.e., via chromosomal amplification or plasmid amplification). Processimprovements are implemented by screening feeding strategies (e.g.,batch, fed-batch, continuous, or recycle), reactor configurations,stirring methods (e.g., via impeller, bubble, air lift, packed bed,solid state, or hollow fiber), pH control, foam, and temperature.Enzymes produced by exemplary large-scale microbial systems include-various serine proteinases, Zn metalloproteinases, asparticproteinases, isomerases, pectinases, lipases, α-amylase, cellases, andglucomylases.

Uses for Polypeptides

The isolated G. oxydans 2-ketoreductase enzymes, and fragmentsmodifications, and variants thereof, are useful for generating chiralalcohols for various biosynthetic or pharmaceutical applications. Invarious aspects, G. oxydans 2-ketoreductase can be used in reductionreactions involving ketones, especially alkylketones (e.g., 2-pentanone,2-heptanone, 2-octanone, 2-decanone, and 2-hexanone. Such reactions canbe used to produce, for example, 2-pentanol, 2-heptanol, 2-octanol,2-decanol, and 2-hexanol. These chiral alcohols can be used asintermediates in synthetic reactions known in the art. In addition,2-ketoreductase enzymes can be used in the synthesis of polyketides inconjunction with other enzymes, including dehydratase, acyl carrierprotein, enoylreductase, 2-ketoacyl ACP synthase, and acyltransferase(see, e.g., M. McPherson et al., 1998, J. Am. Chem. Soc. 120, 3267-3268;U.S. Pat. No. 6,274,560 to Khosla et al.; U.S. Pat. No. 5,962,290 toKhosla et al.; U.S. Pat. No. 6,258,566 to Barr et al.).

In preferred embodiments, the products of reactions G. oxydans2-ketoreductase are useful as intermediates for the synthesis oftherapeutics or other beneficial compounds. For example, polyketides areuseful as a large and diverse class of pharmaceutical products,including antibiotics (e.g., anthracyclines, tetracyclines, polyethers,ansamycins, macrolides of different types, such as polyenes andavermectins as well as classical macrolides such as erythromycins),anti-cancer agents (e.g., mithramycin, daunomycin, and dynemycin A),antifungals (e.g., griseofulvin and strobilurins), antiparasitics (e.g.,avermectin and monensin), immunosuppressive agents (e.g., FK506 andrapamycin), cholesterol-lowering agents (lovastatin and squalestatins),and veterinary products (e.g., monensin and avermectin).

For use in medical or industrial applications, G. oxydans2-ketoreductase enzymes, fragments, modifications, or variants thereofcan be added to a particular chemical reaction by any available means.For example, 2-ketoreductase isolated from natural (e.g., Gluconobacteroxydans cells), recombinant, or synthetic sources may be used.Alternatively, cell extracts or whole cells expressing a secreted formof G. oxydans 2-ketoreductase may be used. Different sources of G.oxydans 2-ketoreductase can be compared to determine the enzyme sourcethat results in, for example, the highest yields of product or thelowest production costs.

Antibodies

Another aspect of the invention pertains to antibodies directed to G.oxydans 2-ketoreductase polypeptides, or fragments or variants thereofThe invention provides polyclonal and monoclonal antibodies that bindketoreductase or ketoreductase fragments. The antibodies may be elicitedin an animal host (e.g., non-human mammal) by immunization with enzymecomponents. Antibodies may also be elicited by in vitro immunization(sensitization) of immune cells. The immunogenic components used toelicit the production of antibodies may be isolated from cells orchemically synthesized. The antibodies may also be produced inrecombinant systems programmed with appropriate antibody-encoding DNA.Alternatively, the antibodies may be constructed by biochemicalreconstitution of purified heavy and light chains. The antibodiesinclude hybrid antibodies, chimeric antibodies, and univalentantibodies. Also included are Fab fragments, including Fab₁ and Fab(ab)₂fragments of antibodies.

In accordance with the present invention, antibodies are directed to aG. oxydans 2-ketoreductase polypeptide (e.g., SEQ ID NO:2), or variants,or fragments thereof. For example, antibodies can be produced to bind toG. oxydans 2-ketoreductase polypeptide encoded by an alternate splicevariant or SNP variant of SEQ ID NO:1. An isolated G. oxydans2-ketoreductase (e.g., SEQ ID NO:2), or variant, or fragment thereof,can be used as an immunogen to generate antibodies using standardtechniques for polyclonal and monoclonal antibody preparation. Afull-length G. oxydans 2-ketoreductase polypeptide can be used or,alternatively, the invention provides antigenic peptide portions of thepolypeptide for use as immunogens. An antigenic peptide comprises atleast 5 contiguous amino acid residues, preferably at least 12contiguous amino acid residues, of the amino acid sequence shown in SEQID NO:2, or a variant thereof, and encompasses an epitope of a G.oxydans 2-ketoreductase polypeptide such that an antibody raised againstthe peptide forms a specific immune complex with a G. oxydans2-ketoreductase sequence.

An appropriate immunogenic preparation can contain, for example,recombinantly produced G. oxydans 2-ketoreductase polypeptide or achemically synthesized polypeptide, or fragments thereof. Thepreparation can further include an adjuvant, such as Freund's completeor incomplete adjuvant, or similar immunostimulatory agent. A number ofadjuvants are known and used by those skilled in the art. Non-limitingexamples of suitable adjuvants include incomplete Freund's adjuvant,mineral gels such as alum, aluminum phosphate, aluminum hydroxide,aluminum silica, and surface-active substances such as lysolecithin,pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpethemocyanin, and dinitrophenol. Further examples of adjuvants includeN-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP),N-acetyl-nor-muramyl-L-alanyl-D-isoglutamine (CGP 11637, referred to asnor-MDP),N-acetylmuramyl-Lalanyl-D-isoglutaminyl-L-alanine-2-(1′-2′-dipalmitoyl-sn-glycero-3hydroxyphosphoryl-oxy)-ethylamine (CGP 19835A, referred to as MTP-PE),and RIBI, which contains three components extracted from bacteria,monophosphoryl lipid A, trehalose dimycolate and cell wall skeleton(MPL+TDM+CWS) in a 2% squalene/Tween 80 emulsion. A particularly usefuladjuvant comprises 5% (wt/vol) squalene, 2.5% Pluronic L121 polymer and0.2% polysorbate in phosphate buffered saline (Kwak et al., 1992, NewEng. J. Med. 327:1209-1215). Preferred adjuvants include complete BCG,Detox, (RIBI, Immunochem Research Inc.), ISCOMS, and aluminum hydroxideadjuvant (Superphos, Biosector). The effectiveness of an adjuvant may bedetermined by measuring the amount of antibodies directed against theimmunogenic peptide.

Polyclonal antibodies to G. oxydans 2-ketoreductase polypeptides orpeptides can be prepared as described above by immunizing a suitablesubject (e.g., horse, donkey, goat, rabbit, rat, mouse, chicken, orother non-human animal) with a ketoreductase immunogen. The antibodytiter in the immunized subject can be monitored over time by standardtechniques, such as with an enzyme linked immunosorbent assay (ELISA)using immobilized G. oxydans 2-ketoreductase polypeptide or peptide. Ifdesired, the antibody molecules can be isolated from the mammal (e.g.,from the blood) and further purified by well-known techniques, such asprotein A chromatography to obtain the IgG fraction.

At an appropriate time after immunization, e.g., when the antibodytiters are highest, antibody-producing cells can be obtained from thesubject and used to prepare monoclonal antibodies by standardtechniques, such as the hybridoma technique (see Kohler and Milstein,1975, Nature 256:495497; Brown et al., 1981, J. Immunol. 127:539-46;Brown et al., 1980, J. Biol. Chem. 255:4980-83; Yeh et al., 1976, PNAS76:2927-31; and Yeh et al., 1982, Int J. Cancer 29:269-75), the human Bcell hybridoma technique (Kozbor et al., 1983, Immunol. Today 4:72), theEBV-hybridoma technique (Cole et al., 1985, Monoclonal Antibodies andCancer Therapy, Alan R. Liss, Inc., pp. 77-96) or trioma techniques.

The technology for producing hybridomas is well-known (see generally R.H. Kenneth, 1980, Monoclonal Antibodies: A New Dimension In BiologicalAnalyses, Plenum Publishing Corp., New York, N.Y.; E. A. Lerner, 1981,Yale J. Biol. Med., 54:387-402; M. L. Gefter et al., 1977, Somatic CellGenet. 3:231-36). In general, an immortal cell line (typically amyeloma) is fused to lymphocytes (typically splenocytes) from a mammalimmunized with a G. oxydans 2-ketoreductase immunogen as describedabove, and the culture supernatants of the resulting hybridoma cells arescreened to identify a hybridoma producing a monoclonal antibody thatbinds G. oxydans 2-ketoreductase polypeptides or peptides.

Any of the many well known protocols used for fusing lymphocytes andimmortalized cell lines can be applied for the purpose of generating anmonoclonal antibody to a G. oxydans 2-ketoreductase (see, e.g., G.Galfre et al., 1977, Nature 266:55052; Gefter et al., 1977; Lerner,1981; Kenneth, 1980). Moreover, the ordinarily skilled worker willappreciate that there are many variations of such methods. Typically,the immortal cell line (e.g., a myeloma cell line) is derived from thesame mammalian species as the lymphocytes. For example, murinehybridomas can be made by fusing lymphocytes from a mouse immunized withan immunogenic preparation of the present invention with an immortalizedmouse cell line. Preferred immortal cell lines are mouse myeloma celllines that are sensitive to culture medium containing hypoxanthine,aminopterin, and thymidine (HAT medium). Any of a number of myeloma celllines can be used as a fusion partner according to standard techniques,e.g., the P3-NS1/1-Ag4-1, P3-x63-Ag8.653, or Sp2/O-Ag14 myeloma lines.These myeloma lines are available from ATCC (American Type CultureCollection, Manassas, Va.). Typically, HAT-sensitive mouse myeloma cellsare fused to mouse splenocytes using polyethylene glycol (PEG).Hybridoma cells resulting from the fusion arc then selected using HATmedium, which kills unfused and unproductively fused myeloma cells(unfused splenocytes die after several days because they are nottransformed). Hybridoma cells producing a monoclonal antibody of theinvention are detected by screening the hybridoma culture supematantsfor antibodies that bind G. oxydans 2-ketoreductase polypeptides orpeptides, e.g., using a standard ELISA assay.

Alternative to preparing monoclonal antibody-secreting hybridomas, amonoclonal antibody can be identified and isolated by screening arecombinant combinatorial immunoglobulin library (e.g., an antibodyphage display library) with the corresponding G. oxydans 2-ketoreductasepolypeptide to thereby isolate immunoglobulin library members that bindthe polypeptide. Kits for generating and screening phage displaylibraries are commercially available (e.g., the Pharmacia RecombinantPhage Antibody System; and the Stratagene SurfZAP™ Phage Display Kit).

Additionally, examples of methods and reagents particularly amenable foruse in generating and screening antibody display library can be foundin, for example, Ladner et al. U.S. Pat. No. 5,223,409; Kang et al. PCTInternational Publication No. WO 92/18619; Dower et al. PCTInternational Publication No. WO 91/17271; Winter et al. PCTInternational Publication WO 92/20791; Markland et al. PCT InternationalPublication No. WO 92/15679; Breitling et al. PCT InternationalPublication WO 93/01288; McCafferty et al. PCT International PublicationNo. WO 92/01047; Garrard et al. PCT International Publication No. WO92/09690; Ladner et al. PCT International Publication No. WO 90/02809;Fuchs et al., 1991, Bio/Technology 9:1370-1372; Hay et al., 1992, Hum.Antibod. Hybridomas 3:81-85; Huse et al., 1989, Science 246:1275-1281;Griffiths et al., 1993, EMBO J 12:725-734; Hawkins et al., 1992, J. Mol.Biol. 226:889-896; Clarkson et al., 1991, Nature 352:624-628; Gram etal., 1992, PNAS 89:3576-3580; Garrad et al., 1991, Bio/Technology9:1373-1377; Hoogenboom et al., 1991, Nuc. Acid Res. 19:4133-4137;Barbas et al., 1991, PNAS 88:7978-7982; and McCafferty et al., 1990,Nature 348:552-55.

Additionally, recombinant antibodies to a G. oxydans 2-ketoreductasepolypeptide, such as chimeric monoclonal antibodies, can be made usingstandard recombinant DNA techniques. Such chimeric monoclonal antibodiescan be produced by recombinant DNA techniques known in the art, forexample using methods described in Robinson et al. InternationalApplication No. PCT/US86/02269; Akira, et al. European PatentApplication 184,187; Taniguchi, M., European Patent Application 171,496;Morrison et al. European Patent Application 173,494; Neuberger et al.PCT International Publication No. WO 86/01533; Cabilly et al. U.S. Pat.No. 4,816,567; Cabilly et al. European Patent Application 125,023;Better et al., 1988, Science 240:1041-1043; Liu et al., 1987, PNAS84:3439-3443; Liu et al., 1987, J. Immunol. 139:3521-3526; Sun et al.,1987, PNAS 84:214-218; Nishimura et al., 1987, Canc. Res. 47:999-1005;Wood et al., 1985, Nature 314:446-449; and Shaw et al., 1988, J. Natl.Cancer Inst. 80:1553-1559; S. L. Morrison, 1985, Science 229:1202-1207;Oi et al., 1986, BioTechniques 4:214; Winter U.S. Pat. No. 5,225,539;Jones et al., 1986, Nature 321:552-525; Verhoeyan et al., 1988, Science239:1534; and Bcidler et al., 1988, J. Immunol. 141:4053-4060.

An antibody against a G. oxydans 2-ketoreductase (e.g., monoclonalantibody) can be used to isolate the corresponding enzyme or enzymefragment by standard techniques, such as affinity chromatography orimmunoprecipitation. For example, antibodies can facilitate thepurification of a natural G. oxydans 2-ketoreductase from cells and of arecombinantly produced G. oxydans 2-ketoreductase or enzyme fragmentexpressed in host cells. In addition, an antibody that binds to a G.oxydans 2-ketoreductase polypeptide can be used to detect thecorresponding enzyme (e.g., in a cell, cellular lysate, or cellsupernatant) in order to evaluate the abundance, localization, orpattern of expression of the protein. Detection methods employingantibodies include well-established techniques, such as Western blot,dot blot, colony blot, ELISA, immunocytochemical, andimmunohistochemical analysis.

Modulators

The G. oxydans 2-ketoreductase, polynucleotides, variants, or fragmentsthereof, can be used to screen for test agents (e.g., agonists,antagonists, or inhibitors) that modulate the levels or activity of thecorresponding enzyme. In addition, these ketoreductase molecules can beused to. identify endogenous modulators that bind to polypeptides orpolynucleotides in the G. oxydans cell. In one aspect of the presentinvention, the full-length G. oxydans 2-ketoreductase (e.g., SEQ IDNO:2) is used to identify modulators. Alternatively, variants orfragments of a G. oxydans 2-ketoreductase are used. Such fragments maycomprise, for example, one or more domains of a G. oxydans2-ketoreductase (e.g., the short chain dehydrogenase domain) disclosedherein. A wide variety of assays may be used for these screens,including in vitro protein-protein binding assays, electrophoreticmobility shift assays, immunoassays, and the like.

The term “modulator” as used herein describes any test agent, molecule,protein, peptide, or compound with the capability of directly orindirectly altering the physiological function, stability, or levels ofG. oxydans 2-ketoreductase. Modulators that bind to the G. oxydans2-ketoreductase polypeptides or polynucleotides of the invention arepotentially useful in biotechnology or pharmaceutical applications, asdescribed in detail herein. Test agents useful as modulators mayencompass numerous chemical classes, though typically they are organicmolecules, preferably small organic compounds having a molecular weightof more than 50 and less than about 2,500 daltons. Such molecules cancomprise functional groups necessary for structural interaction withproteins, particularly hydrogen bonding, and typically include at leastan amine, carbonyl, hydroxyl or carboxyl group, preferably at least twoof the functional chemical groups. Test agents which can be used asmodulators often comprise cyclical carbon or heterocyclic structuresand/or aromatic or polyaromatic structures substituted with one or moreof the above functional groups. Test agents can also comprisebiomolecules including peptides, saccharides, fatty acids, steroids,purines, pyrimidines, derivatives, structural analogs, or combinationsthereof.

Test agents finding use as modulators may include, for example, 1)peptides such as soluble peptides, including Ig-tailed fusion peptidesand members of random peptide libraries (see, e.g., Lam et al., 1991,Nature 354:82-84; Houghten et al., 1991, Nature 354:84-86) andcombinatorial chemistry-derived molecular libraries made of D- and/orL-configuration amino acids; 2) phosphopeptides (e.g., members of randomand partially degenerate, directed phosphopeptide libraries, see, e.g.,Songyang et al, (1993) Cell 72:767-778); 3) antibodies (e.g.,polyclonal, monoclonal, humanized, anti-idiotypic, chimeric, and singlechain antibodies as well as Fab, F(ab′)₂, Fab expression libraryfragments, and epitope-binding fragments of antibodies); and 4) smallorganic and inorganic molecules.

Test agents and modulators can be obtained from a wide variety ofsources including libraries of synthetic or natural compounds. Syntheticcompound libraries are commercially available from, for example,Maybridge Chemical Co. (Trevillet, Comwall, UK), Comgenex (Princeton,N.J.), Brandon Associates (Merrimack, N.H.), and Microsource (NewMilford, Conn.). A rare chemical library is available from AldrichChemical Company, Inc. (Milwaukee, Wis.). Natural compound librariescomprising bacterial, fungal, plant or animal extracts are available.from, for example, Pan Laboratories (Bothell, Wash.). In addition,numerous means are available for random and directed synthesis of a widevariety of organic compounds and biomolecules, including expression ofrandomized oligonucleotides.

Alternatively, libraries of natural compounds in the form of bacterial,fungal, plant and animal extracts can be readily produced. Methods forthe synthesis of molecular libraries are readily available (see, e.g.,DeWitt et al., 1993, Proc. Natl. Acad. Sci. USA 90:6909; Erb et al.,1994, Proc. Natl. Acad. Sci. USA 91:11422; Zuckermann et al., 1994, J.Med. Chem. 37:2678; Cho et al., 1993, Science 261:1303; Carell et al.,1994, Angew. Chem. Int. Ed. Engl. 33:2059; Carell et al., 1994, Angew.Chem. Int. Ed. Engl. 33:2061; and in Gallop et al., 1994, J. Med. Chem.37:1233). In addition, natural or synthetic compound libraries andcompounds can be readily modified through conventional chemical,physical and biochemical means (see, e.g., Blondelle et al., 1996,Trends in Biotech. 14:60), and may be used to produce combinatoriallibraries. In another approach, previously identified pharmacologicalagents can be subjected to directed or random chemical modifications,such as acylation, alkylation, esterification, amidification, and theanalogs can be screened for 2-ketoreductase-modulating activity.

Numerous methods for producing combinatorial libraries are known in theart, including those involving biological libraries; spatiallyaddressable parallel solid phase or solution phase libraries; syntheticlibrary methods requiring deconvolution; the ‘one-bead one-compound’library method; and synthetic library methods using affinitychromatography selection. The biological library approach is limited topolypeptide libraries, while the other four approaches are applicable topolypeptide, non-peptide oligomer, or small molecule libraries ofcompounds (K. S. Lam, 1997, Anticancer Drug Des. 12:145).

Libraries may be screened in solution (e.g., Houghten, 1992,Biotechniques 13:412-421), or on beads (Lam, 1991 Nature 354:82-84),chips (Fodor, 1993 Nature 364:555-556), bacteria or spores (Ladner U.S.Pat. No. 5,223,409), plasmids (Cull et al., 1992 Proc. Natl. Acad. Sci.USA 89:1865-1869), or on phage (Scott and Smith, 1990, Science249:386-390; Deviun, 1990, Science 249:404-406; Cwirla et al., 1990,Proc. Natl. Acad. Sci. USA 97:6378-6382; Felici, 1991, J. Mol. Biol.222:301-310; Ladner, supra).

Where the screening assay is a binding assay, a G. oxydans2-ketoreductase polypeptide, polynucleotide, functional equivalent, orfragment thereof, may be joined to a label, where the label can directlyor indirectly provide a detectable signal. Various labels includeradioisotopes, fluorescers, chemiluminescers, enzymes, specific bindingmolecules, particles, e.g. magnetic particles, and the like. Preferredfluorescent labels include, for example, Cy3, Cy5, GFP (e.g., EGFP,DsRed, dEFP, etc. (CLONTECH, Palo Alto, Calif.)), Alexa, BODIPY,fluorescein (e.g., FluorX, DTAF, and FITC), rhodamine (e.g., TRITC),auramine, Texas Red, AMCA blue, and Lucifer Yellow. Preferred isotopelabels include ³H, ¹⁴C, ³²P, ³⁵S, ³⁶Cl, ⁵¹Cr, ⁵⁷Co, ⁵⁸Co ⁵⁹Fe, ⁹⁰Y,¹²⁵I, ¹³¹I, and ¹⁸⁶Re.

Non-limiting examples of enzyme labels include peroxidase,β-glucuronidase, β-D-glucosidase, β-D-galactosidase, urease, glucoseoxidase plus peroxidase, and alkaline phosphatase (see, e.g., U.S. Pat.Nos. 3,654,090; 3,850,752 and 4,016,043). Enzymes can be conjugated byreaction with bridging molecules such as carbodiimides, diisocyanates,glutaraldehyde, and the like. Enzyme labels can be detected visually, ormeasured by calorimetric, spectrophotometric, fluorospectrophotometric,amperometric, or gasometric techniques. Other labeling systems, such asavidin/biotin, Tyramide Signal Amplification (TSA™), anddigoxin/anti-digoxin, are known in the art, and are commerciallyavailable (see, e.g., ABC kit, Vector Laboratories, Inc., Burlingame,Calif.; NEN® Life Science Products, Inc., Boston, Mass.). For thespecific binding members, the complementary member would normally belabeled with a molecule that provides for detection, in accordance withknown procedures.

A variety of other reagents may be included in the screening assay.These include reagents like salts, neutral proteins, e.g. albumin,detergents, etc., that are used to facilitate optimal protein-proteinbinding and/or reduce non-specific or background interactions. Reagentsthat improve the efficiency of the assay, such as protease inhibitors,nuclease inhibitors, anti-microbial agents, etc., may be used. Thecomponents are added in any order that produces the requisite binding.Incubations are performed at any temperature that facilitates optimalactivity, typically between 4° and 40° C. Incubation periods areselected for optimum activity, but may also be optimized to facilitaterapid high-throughput screening. Normally, between 0.1 and 1 hr will besufficient. In general, a plurality of assay mixtures is run in parallelwith different agent concentrations to obtain a differential response tothese concentrations. Typically, one of these concentrations serves as anegative control, i.e. at zero concentration or below the level ofdetection.

To perform cell-free screening assays, it may be desirable to immobilizeeither the a G. oxydans 2-ketoreductase polypeptide, polynucleotide,variant, or fragment to a surface to facilitate identification ofmodulators that bind to these molecules, as well as to accommodateautomation of the assay. For example, a fusion protein comprising a G.oxydans 2-ketoreductase polypeptide and an affinity-tag can be producedas described in detail herein. In one embodiment, a GST-fusion proteincomprising a G. oxydans 2-ketoreductase polypeptide is adsorbed ontoglutathione sepharose beads (Sigma Chemical, St. Louis, Mo.) orglutathione-derivatized microtiter plates. Cell lysates (e.g.,containing ³⁵S labeled polypeptides) are added to the polypeptide-coatedbeads under conditions to allow complex formation (e.g., atphysiological conditions for salt and pH). Following incubation, thepolypeptide-coated beads are washed to remove any unbound polypeptides,and the amount of immobilized radiolabel is determined. Alternatively,the complex is dissociated and the radiolabel present in the supematantis determined. In another approach, the beads are analyzed by SDS-PAGEto identify ketoreductase-binding polypeptides.

Various binding assays can be used to identify modulators that alter thefunction or levels of G. oxydans 2-ketoreductase. Such assays aredesigned to detect the interaction. of test agents with G. oxydans2-ketoreductase polypeptides, polynucleotides, variants, or fragmentsthereof. Interactions may be detected by direct measurement of binding.Non-limiting examples of useful binding assays are detailed as follows.Modulators that bind to G. oxydans 2-ketoreductase polypeptides,polynucleotides, functional equivalents, or fragments thereof, can beidentified using real-time Bimolecular Interaction Analysis (BIA;Sjolander et al., 1991, Anal. Chem. 63:2338-2345; Szabo et al., 1995,Curr. Opin. Struct. Biol. 5:699-705; e.g., BIAcore™; LKB Pharmacia,Sweden). Modulators can also be identified by scintillation proximityassays (SPA, described in U.S. Pat. No. 4,568,649). Binding assays usingmitochondrial targeting signals (Hurt et al., 1985, EMBO J. 4:2061-2068;Eilers and Schatz, 1986, Nature 322:228-231) a plurality of definedpolymers synthesized on a solid substrate (Fodor et al., 1991, Science251:767-773) may also be employed.

Two-hybrid systems may be used to identify modulators (see, e.g., U.S.Pat. No. 5,283,317; Zervos et al., 1993, Cell 72:223-232; Madura et al.,1993, J. Biol. Chem. 268:12046-12054; Bartel et al., 1993, Biotechniques14:920-924; Iwabuchi et al., 1993, Oncogene 8:1693-1696; and Brent WO94/10300). Alternatively, three-hybrid (Licitra et al., 1996, Proc.Natl. Acad. Sci. USA 93:12817-12821), and reverse two-hybrid (Vidal etal., 1996, Proc. Natl. Acad. Sci. USA 93:10315-10320) systems may beused. Commercially available two-hybrid systems such as the CLONTECHMatchmaker™ systems and protocols (CLONTECH Laboratories, Inc., PaloAlto, Calif.) are also useful (see also, A. R. Mendelsohn et al., 1994,Curr. Op. Biotech. 5:482; E. M. Phizicky et al., 1995, MicrobiologicalRev. 59:94; M. Yang et al., 1995, Nucleic Acids Res. 23:1152; S. Fieldset al., 1994, Trends Genet. 10:286; and U.S. Pat. Nos. 6,283,173 and5,468,614).

Several methods of automated assays have been developed in recent yearsso as to permit screening of tens of thousands of test agents in a shortperiod of time. High-throughput screening methods are particularlypreferred for use with the present invention. The binding assaysdescribed herein can be adapted for high-throughput screens, oralternative screens may be employed. For example, continuous format highthroughput screens (CF-HTS) using at least one porous matrix allows theresearcher to test large numbers of test agents for a wide range ofbiological or biochemical activity (see U.S. Pat. No. 5,976,813 toBeutel et al.). Moreover, CF-HTS can be used to perform multi-stepassays.

Alternatively, interactions with test agents may be detected by indirectindicators of binding, such as stabilization/destabilization of proteinstructure, or activation/inhibition of biological function. For example,modulating agents may be identified by an increase or decrease in levelsof chiral alcohol produced by G. oxydans 2-ketoreductase upon incubationwith substrate. Specifically, agonist agents would be expected toincrease alcohol levels, whereas antagonist agents would be expected todecrease alcohol levels produced by the enzyme.

In one embodiment of the present invention, an agonist or antagonist isidentified by incubating the disclosed G. oxydans 2-ketoreductase, orfragments or variants thereof, with a test agent. The G. oxydans2-ketoreductase may be expressed by host cells, or may be isolatedtherefrom. The G. oxydans 2-ketoreductase and test agent is incubatedwith substrate, and levels of alcohol product are determined andcompared with standard levels. Increased levels of alcohol indicateidentification of an agonist agent, while decreased levels of alcoholindicate identification of an antagonist agent.

Embodiments

This invention encompasses, but is not limited to, the followingembodiments:

-   -   An isolated nucleic acid comprising a nucleotide sequence        encoding amino acid sequence SEQ ID NO:2.    -   An isolated nucleic acid comprising a nucleotide sequence        encoding at least 12 contiguous residues of amino acid sequence        SEQ ID NO:2.    -   An isolated nucleic acid comprising a nucleotide sequence        encoding at least 12 contiguous residues of the short chain        dehydrogenase domain of amino acid sequence SEQ ID NO:2.    -   An isolated nucleic acid comprising nucleotide sequence SEQ ID        NO:1.    -   An isolated nucleic acid comprising at least 21 contiguous        nucleotides of nucleotide sequence SEQ ID NO:1.    -   An isolated nucleic acid comprising a nucleotide sequence which        is at least 54% identical to nucleotide sequence SEQ ID NO:1.    -   An isolated nucleic acid comprising a nucleotide sequence which        is complementary to a nucleotide sequence of the invention        (above).    -   A vector comprising an isolated nucleic acid of the invention        (above).    -   A host cell comprising a vector of the invention (above),        wherein the host cell is selected from the group consisting of        bacterial, fungal, insect, mammalian, and plant cells.    -   The bacterial host cell of the invention (above), wherein the        bacterial host cell is selected from the group consisting of        Escherichia coli, Staphlococcus aureus, Bacillus licheniformis,        Bacillus amyloliquefaciens, Bacillus subtilis, Streptomyces        lividans, and Streptomyces coelicolor.    -   A probe comprising an isolated nucleic acid of the invention        (above). In specific aspects, the probe may comprise a        nucleotide sequence selected from the group consisting of SEQ ID        NO:9-SEQ ID NO:10.    -   A primer comprising an isolated nucleic acid of the invention        (above). In specific aspects, the primer may comprise a        nucleotide sequence selected from the group consisting of SEQ ID        NO:9-SEQ ID NO:10.    -   An isolated polypeptide (e.g., recombinant polypeptide)        comprising amino acid sequence SEQ ID NO:2.    -   An isolated polypeptide (e.g., recombinant polypeptide)        comprising at least 12 contiguous residues of amino acid        sequence SEQ ID NO:2.    -   An isolated polypeptide (e.g., recombinant polypeptide)        comprising at least 12 contiguous residues of the short chain        dehydrogenase domain of amino acid sequence SEQ ID NO:2.    -   An isolated polypeptide (e.g., recombinant polypeptide)        comprising an amino acid sequence which is at least 54%        identical to amino acid sequence SEQ ID NO:2.    -   An antibody which binds to an isolated polypeptide of the        invention (above). In one aspect, the antibody may be        monoclonal.    -   A kit for detecting a nucleic acid comprising:        -   a) the a probe of the invention (above); and        -   b) at least one component to detect binding of the probe to            a nucleic acid.    -   A kit for detecting an amino acid sequence comprising:        -   a) an antibody of the invention (above); and        -   b) at least one component to detect binding of the antibody            to an amino acid sequence.    -   A method for detecting a nucleic acid comprising:        -   a) incubating a probe of the invention (above) with a            biological sample comprising nucleic acids, thereby forming            a hybridization complex; and        -   b) detecting the complex formed in (a), wherein the presence            of the complex indicates detection of a nucleic acid.    -   A method for detecting a polypeptide comprising:        -   a) incubating an antibody of the invention (above) with a            biological sample comprising polypeptides, thereby forming a            complex; and        -   b) detecting the complex formed in (a), wherein the presence            of the complex indicates detection of a polypeptide.    -   A method for detecting a binding factor comprising:        -   a) incubating an isolated nucleic acid of the invention            (above) with a test agent, thereby forming a complex; and        -   b) detecting the complex formed in (a), wherein the presence            of the complex indicates detection of a binding factor.    -   A method for detecting a binding factor comprising:        -   a) incubating an isolated polypeptide of the invention            (above) with a test agent, thereby forming a complex; and        -   b) detecting the complex formed in (a), wherein the presence            of the complex indicates detection of a binding factor.    -   A method for producing a recombinant polypeptide comprising:        -   a) culturing a host cell of the invention (above) under            conditions suitable for the production of a recombinant            polypeptide; and        -   b) recovering the recombinant polypeptide from the host cell            or host cell culture, thereby producing the recombinant            polypeptide.    -   A method for producing a recombinant polypeptide comprising:        -   a) culturing a bacterial host cell of the invention (above)            under conditions suitable for the expression of a            recombinant polypeptide; and        -   b) recovering the recombinant polypeptide from the host cell            or host cell culture, thereby producing the recombinant            polypeptide.    -   A method of isolating a recombinant polypeptide comprising:        -   a) incubating a biological sample obtained from a host cell            expressing recombinant polypeptide comprising amino acid            sequence SEQ ID NO:2 with an antibody of the invention            (above), thereby forming a complex; and        -   b) recovering a polypeptide from the complex, thereby            isolating the polypeptide.    -   A method of producing a chiral alcohol comprising: incubating an        isolated polypeptide of the invention (above) with a ketone        substrate under conditions to allow reduction of the ketone        substrate, thereby producing a chiral alcohol. In one aspect,        the ketone substrate is an alkylketone. In various aspects, the        alkylketone is selected from the group consisting of        2-pentanone, 2-heptanone, 2-octanone, 2-decanone, and        2-hexanone.    -   A method for detecting an agonist agent comprising:        -   a) incubating a host cell of the invention (above), with a            test agent and a ketone substrate under conditions to allow            reduction of the ketone substrate and production of alcohol;        -   b) measuring levels of alcohol, produced in step (a); and        -   c) comparing the levels determined in step (b) to levels            produced in the absence of the test agent, wherein an            increase in levels indicates detection of an agonist agent.            In one aspect, the ketone substrate is an alkylketone. In            various aspects, the alkylketone is selected from the group            consisting of 2-pentanone, 2-heptanone, 2-octanone,            2-decanone, and 2-hexanone.    -   A method for detecting an agonist agent comprising:        -   a) incubating a bacterial host cell of the invention (above)            with a test agent and a ketone substrate under conditions to            allow reduction of the ketone substrate and production of            alcohol;        -   b) measuring levels of alcohol produced in step (a); and        -   c) comparing the levels determined in step (b) to levels            produced in the absence of the test agent, wherein an            increase in levels indicates detection of an agonist agent.            In one aspect, the ketone substrate is an alkylketone. In            various aspects, the alkylketone is selected from the group            consisting of 2-pentanone, 2-heptanone, 2-octanone,            2-decanone, and 2-hexanone.    -   A method for detecting an agonist agent comprising:        -   a) incubating an isolated polypeptide of the invention            (above) with a test agent and a ketone substrate under            conditions to allow reduction of the ketone substrate and            production of alcohol;        -   b) measuring levels of alcohol produced in step (a); and        -   c) comparing the levels determined in step (b) to levels            produced in the absence of the test agent, wherein an            increase in levels indicates detection of an agonist agent.            In one aspect, the ketone substrate is an alkylketone. In            various aspects, the alkylketone is selected from the group            consisting of 2-pentanone, 2-heptanone, 2-octanone,            2-decanone, and 2-hexanone.    -   An American Type Culture Collection deposit deposited as ATCC        Accession No. PTA-3864.    -   A nucleic acid consisting of the nucleotide sequence deposited        as ATCC Accession No. PTA-3864.    -   A recombinant polypeptide encoded by the nucleotide sequence        deposited as ATCC Accession No. PTA-3864.

EXAMPLES

The examples as set forth herein are meant to exemplify the variousaspects of the present invention and are not intended to limit theinvention in any way.

Example 1 Purification of G. Oxydans 2-Ketoreductase

Fermentation: Gluconobacter oxydans (SC13851) was grown on aglycerol-containing medium as follows. Cultures were grown in 500 mlErlenmeyer flasks for 24 hr in 100 ml medium A (5% glycerol, 0.5% yeastextract, 0.05% ammonium sulfate, 0.3% peptone, 0.05% K₂HPO₄, 0.02%MgSO₄.7H₂O, 0.001% NaCl, 0.001% FeSO₄.7H₂O, and 0.001% MnSO₄.7H₂O).After 24 hr, the flask cultures were used to inoculate (1% v/v inoculum)a 15 L fermentor containing medium A. The fermentation was carried outat 28° C. for 24 hr. A 4000 L fermentor (Expend Industries, Inc.,Brooklyn, N.Y.) was inoculated with 10 L inoculum from the 15 Lfermentor. The 4000 L fermentor contained medium A with 0.05% antifoamSAG 5693. The fermentor was operated at 28° C., 100 LPM airflow, 690mbar pressure, and 620 rpm agitation for 48 hr.

Cell recovery: The fermentor broth was cooled to 8° C. at the harvest.The tank was pressurized to 15 psig and broth was diverted to a Sharples(Alfa Laval Separation, Inc., Warminster, Pa.) centrifuge running at18,000×g. The broth was processed at 3.2 L/min and recovered cells werestored at −70° C. until further use.

Purification of 2-ketoreductase: All the purification steps were carriedout at 4° C. Forty-four grams of cells were suspended in 0.3 L buffer A(50 mM Tris-HCl, pH 7.5, 1 mM CaCl₂, and 1 mM MgCl₂). After 30 min ofhomogenization, the cell suspension was passed through a microfluidizer(Microfludics International Corporation, Newton, Mass.) twice at 12,000psi. The supematant obtained by centrifugation (at 30,000×g for 30 min)was loaded onto DEAE cellulose column (400 ml) (Whatman, Maidstone,England), which was pre-equilibrated with buffer A. The enzyme activitywas eluted with a 0 to 0.8 M NaCl gradient in buffer A.

The active fractions were pooled, and ammonium sulfate at 132 g/L wasadded before loading onto a phenylsepharose column (350 ml), which waspre-equilibrated with buffer A containing 1 M ammonium sulfate. Thecolumn was then washed with buffer A containing 1 M ammonium sulfate andthe enzyme was eluted with a 1 M to 0 M ammonium sulfate gradient (totalvolume, 1.2 L). The fractions containing the active enzyme were pooled(150 ml) and concentrated with an Amicon YM-30 membrane (Amicon,Beverly, Mass.) to 8 ml.

The enzyme was then loaded onto a Sephacryl S-200 gel-filtration column(400 ml column) (Pharmacia, Piscataway, N.J.). The enzyme-was elutedwith buffer A containing 0.1 M NaCl with a flow rate of 0.8 ml/min. Theactive fractions from the gel-filtration column were pooled, and thenloaded onto a mono Q ion-exchange (BioRad Q2) column (Bio-Rad, Hercules,Calif.). The enzyme activity was eluted with a 0 to 0.8 M NaCl gradientin buffer A.

The fractions containing the active enzyme were pooled (5.6 ml) andconcentrated with a Centricon-30 membrane to 0.6 ml. The enzyme was thenloaded onto a Superdex-75 gel-filtration column (FPLC; Pharmacia) andeluted with buffer A containing 0.1 M NaCl. The enzyme present infraction 14 was analyzed by sodium dodecyl sulfate-polyacrylamide(SDS-PAGE) electrophoresis. This analysis indicated that the enzyme waspresent as a single band on the gel with a calculated molecular weightof 29 kilodaltons.

Example 2 Analysis of Purified G. Oxydans 2-Ketoreductase

Protein assay: The Bio-Rad protein assay was used to determine proteinconcentration. The assay was performed according to the manufacturer'sprotocol (Bio-Rad). Samples containing 1-10 μl of enzyme fraction werebrought to a volume of 0.8 ml with water. Next, 0.2 ml of the Bio-Radreagent was added to the 0.8 ml sample solution. This was mixedthoroughly. The absorbance of the solution was measured at 595 nm. Theprotein concentration (mg/ml) was calculated from the standard curveusing bovine serum albumin as standard protein.

Enzyme activity units: One unit (U) of enzyme activity was defined asone micromole of S-2-pentanol formed in 1 hr under the conditionsdescribed above. Results from the protein analysis of G. oxydans2-ketoreductase are summarized below. TABLE 1 Enzyme Activity Sp.Activity S-2-Pentanol Steps Volume (mL) (Units) Protein (mg) (Units/mg)(e e) Purification Fold Cell extract 300 390.00 729.00 0.50 1.00 DEAECellulose 180 235.80 185.40 1.27 25.40 Phenylsepharose 150 186.00 72.002.58 51.60 Amicon concentration 10 Sephacryl S200 Gel filtratio 22 27.284.40 6.20 >99 124.00 Mono Q column 5.6 35.39 3.64 9.72 194.40 Centriconconcentration 0.25 Sephadex-75 Gel filtration 0.75 13.28 0.90 14.76 >99295.20

2-Pentanone reduction assays were carried out using both resting cellsand the cell extract (soluble enzyme) of Gluconobacter oxydans.

Whole cell assays: Three grams of wet cell paste were suspended in 15 mlbuffer containing 0.1 M Tris-HCl, pH 8, and 5 mM EDTA. The cellsuspension was treated with 0.36 ml toluene. The treated cell suspensionwas shaken gently for 30 min in a 50 ml Erlenmeyer flask. The cells werethen collected by centrifuging at 18,000×g for 20 minutes. Toluenetreated cells (0.25 g) were suspended in 10 ml of 0.2 M Tris-HCl bufferpH 7.5, containing 10 mM CaCl₂ and 10 mM MgCl₂ in a 25 ml Teflon® flask.The following components were added to the reaction mixture: 7 mg NAD⁺,0.136 g sodium formate, 1.5 U formate dehydrogenase, and 0.025 ml2-pentanone (Sigma,. St. Louis, Mo.). The reaction mixture in the flaskwas incubated in a shaker at 28° C. with agitation at 200 rpm. Atvarious time points (2-18 hr), samples containing 0.5 ml of reactionmixture were removed, and 2 ml of ethyl acetate was added to each sampleto stop the reaction. The organic layer was separated by centrifugationand was used to analyze both the substrate and product.

Soluble enzyme assays: Samples of the enzyme were incubated in areaction mixture (5 ml) containing 0.35 mg NAD⁺, 68 mg sodium formate,0.75 U formate dehydrogenase, and 5 mg 2-pentanone. Reactions werecarried out in a Teflon® flask at 28° C. on a shaker at 200 rpm. After18 hr, the reactions were quenched with 10 ml of ethyl acetate andanalyzed by gas chromatography.

Analysis of enantiomeric alcohols by gas chromatography: Samples wereextracted in ethyl acetate and dried over anhydrous magnesium sulfate.Samples were then applied onto a Astec Chiraldex G-TA, gammacyclodextrin column (20 m×0.25 mm×0.125 μm thickness; Astec, Whippany,N.J.) equipped with a guard column (Hewlett-Packard Ultra II, 5% phenylmethyl silicone, 5 m×0.32 mm×0.17 μm thickness; Agilent Technologies,Palo Alto, Calif.). The temperature of the injector was set at 150° C.and the detector of the chromatograph (Hewlett-Packard 5890) was set at200° C. Detection was carried out with a flame ionization detector(Agilent Technologies). The separation was carried out by a gradientunder the following conditions: 28° C. for 15 minutes, 5OC/min to 50° C.and hold 5 minutes. The helium flow rate was maintained at 22 cm/min.Under these conditions, the retention times for S-2-pentanol,R-2-pentanol, and 2-pentanone were 10.85, 11.67 and 17.84 minutesrespectively.

Peptide Sequencing of the purified 2-ketoreductase: The purified proteinwas sent for N-terminal and internal peptide sequences to ArgoBioAnalytica, Inc., Morris Plains, N.J. The following are the sequencesobtained for the 2-ketoreductase.

N-terminal sequence NH₂-Ser-Leu-Ser-Gly-Lys-Ile-Ala-Ala- (SEQ ID NO:4)Val-Thr-Gly-Ala-Ala-Gln-Gly-COOH.

Internal peptides Peptide 1: NH₂-Lys-Arg-Met-Ala-Glu-Ile-Thr-Gly- (SEQID NO:5) Thr-Glu-Ile-COOH; and Peptide 2:NH₂-Lys-Val-Glu-Ala-Leu-Gly-Arg-Arg- (SEQ ID NO:6) Ala-Val-COOH.

Example 3 Identification of the 2-Ketoreductase Gene

Gluconobacter oxydans (BMS Collection No. SC13851; ATCC No. 621) wasgrown in 50 ml LB medium (1% tryptone, 0.5% yeast extract, 0.5% NaCl) at37° C. for 16 hr at 200 rpm in a shaker. The cells were harvested bycentrifugation and the chromosomal DNA was prepared (see Ausubel et al.(Eds.), 1981, Current Protocols in Molecular Biology, vol. 2, section13.11.2, John Wiley and Sons, New York). Degenerate PCR primers based oninternal peptides (oligo GO1: 5′-MR GTI GAR GCI YTI GGI MGI MGI GCIGT-3′; SEQ ID NO:7) (oligo GO4: 5′-ATY TCI GTI CCI GTI ATY TCI GCCAT-3′; SEQ ID NO:8), where “Y”=C+T; “R”=A+G; “I”=deoxyinosine; and“M”=A+C), were used to amplify the gene using genomic DNA as template.The amplification conditions included incubation at 94° C. for 1 min,followed by 30 cycles at 94° C. for 0.5 min; 50° C. for 0.5 min; and 72°C. for 0.5 min using a Hybaid PCR Express thermocycler (ThermoHybaid US,Franklin, Mass.). The PCR fragments were electrophoresed at 60 V for 2hr through a 0.8% agarose gel in TAE buffer (0.04 M Trizma base, 0.02 Macetic acid, and 0.001 M EDTA, pH 8.3) containing 0.5 μg/ml ethidiumbromide. The 400 bp PCR Fragment was identified by comparison to a 1 kbPlus DNA ladder (Invitrogen) and excised using a scalpel. The DNA wasisolated from the agarose using the QIAquick Gel Extraction Kit (QIAGEN,Chatsworth, Calif.). The resulting 400 base pair (bp) PCR fragment wascloned into pCR2.1™ using the TA Cloning kit (Invitrogen, Carlsbad,Calif.).

To isolate the complete 2-ketoreductase gene, G. oxydans chromosomal DNAwas cleaved with restriction endonucleases BamHI, EcoRI, EcoRV, HindIII,NotI, PstI, SacI, SpaI, XbaI and XhoI under conditions recommended bythe manufacturer (Promega, Madison, Wis.). Approximately 3 μg of eachdigested sample was electrophoresed at 20 v for 18 hr through a 0.8%agarose gel in TAE buffer (0.04 M Trizma base, 0.02 M acetic acid, and0.001 M EDTA, pH 8.3) containing 0.5 μg/ml ethidium bromide. Fragmentswere transferred to a Hybond N+ nylon filter (Amersham Pharmacia,Piscatatway, N.J.) using a VacuGene blotting apparatus (AmershamPharmacia). To identify the 2-ketoreductase gene, the 400 bp fragmentwas obtained by digesting the pCR2.1™ plasmid with EcoRI, labeled usingPCR DIG Probe Kit (Roche Biochemicals, Indianapolis, Ind.), and thelabeled fragment was used as a probe.

Hybridization to the filter containing G. oxydans chromosomal digests,washing, and detection were performed according to materials anddirections supplied with the DIG High Prime DNA Labeling and DetectionStarter Kit II (Roche Biochemicals). Stringent wash conditions carriedout using 1×SSC (20×SSC is 173.5 g NaCl, 88.2 g NaCl, pH 7.0) and 0.1%sodium dodecyl sulfate at 68° C. A single hybridizing fragment wasvisible in all the endonuclease digests. A 4 kb BamHI fragment waschosen for further analysis. Approximately 10 μg of G. oxydanschromosomal DNA was digested using 25 U BamHI for 2 hr at 37° C. in afinal volume of 0.1 ml with the buffer recommended by the manufacturer(Promega). The digested DNA was electrophoresed on a 0.8% agarose gel inTAE buffer at 20 V for 18 hr. Fragments between 3.8 and 4.5 kb wereidentified by comparison to a 1 kb Plus DNA ladder (Invitrogen) andexcised using a scalpel.

The DNA was isolated from the agarose using the QIAquick Gel ExtractionKit (QIAGEN) and ligated to BamHI-cleaved pZero2 (Invitrogen, Carlsbad,Calif.) vector DNA in a 2:1 molar ratio in a total volume of 10 μL at22° C. for 2 hr. Two microliters of ligated DNA was used to transform0.04 ml competent E. coli DH10B cells (Invitrogen) by electroporation.SOC medium was immediately added (0.96 ml; SOC is, per liter, 0.5% yeastextract, 2% tryptone, 10 mM NaCl, 2.5 mM KCl, 10 mM MgCl₂, 10 mM MgSO₄,and 20 mM glucose). Cells incubated in a shaker for 1 hr at 37° C. and225 rpm.

Cells were spread onto a 132 mm Hybond N+ membrane placed on top of LBkanamycin agar medium. Kanamycin was purchased from Sigma Chemical Co.,and used at final concentration of 50 μg/ml. Cells were grown at 37° C.for 20 hr. Colonies were replicated onto two fresh filters placed on topof LB kanamycin agar medium and incubated at 30° C. for 16 hrs. Colonieswere lysed in situ by placing the filters on a piece of Whatman 3MMpaper saturated with 0.5 M NaOH for 5 min. The filters were dried for 5min on Whatman paper, then neutralized on 3MM paper soaked in 1.0 MTris-HCl, pH 7.5 for 2 min, and dried again for 2 min. Membranes wereplaced on top of 3MM paper saturated with 1.0 M Tris-HCl, pH 7.0/1.5 MNaCl for 10 min.

DNA was crosslinked to the filters by exposure to ultraviolet light in aStratagene UV Stratalinker 2400 set to “auto crosslink” mode(Stratagene, LaJolla, Calif.). Cell debris was removed from themembranes by immersing in 3×SSC/0.1% SDS, wiping the surface with awetted Kimwipee, then incubating in the same solution heated to 65° C.for 3 hr with agitation. Filters were rinsed with dH₂O and usedimmediately or wrapped in SaranWrap® and stored at 4° C. Hybridization,washing, and detection was performed as described above using the 400 bpG. oxydans 2-ketoreductase gene probe.

Eight putative hybridizing colonies were picked from the master plate,inoculated into SOC medium containing kanamycin, and grown at 37° C. for24 hr at 250 rpm. These colonies were also tested for the presence of400 bp fragment with GOI1 and GOI4 primers using the conditionsdescribed previously. Six of the eight colonies gave the expected PCR400 bp fragment confirming the BamHI fragment contained at least aportion of the 2-pentanone reductase gene. Cells from one milliliter ofcell culture from two selected. colonies were pelleted bycentrifugation. Plasmid DNA was isolated using the QIAprep SpinMini-plasmid Isolation Kit (QIAGEN). An aliquot of plasmid DNA wasdigested with BamHI to confirm the presence of the 4.0 kb fragment.

Example 4 Sequencing and Sequence Analysis of G. Oxydans 2Ketoreductase-Gene

DNA sequencing of the 4.0 kb insert of 2-ketoreductase gene wasperformed at the Bristol-Myers Squibb sequencing facility. DNAsequencing of the 4.0 kb insert was carried out using the BigDyeterminator kit and DNA sequencing unit model 377 (Applied Biosystems,Foster City, Calif.). The complete 2-ketoreductase nucleotide sequenceand predicted amino acid sequence is shown in FIGS. 1-1 to 1-3. Thecoding region was determined to be 780 bp in length. The nucleotidesequence was determined to encode a 260-amino acid protein (MW=27,220daltons). The G. oxydans 2-ketoreductase amino acid sequence showedsignificant homology to other dehydrogenases including acetoindehydrogenase, L-2,3-butanediol dehydrogenase, sorbitol dehydrogenase,polyketide reductase, and glucose dehydrogenase. In addition, theN-terminus of G. oxydans 2-ketoreductase showed homology to aribitol-dehydrogenase from Klebsiell aerogenes (Loviny et. al., 1985,Biochem. J. 230:579-585).

A conserved domain search (CD-Search; http://www.ncbi.nlm.nih.gov/blast/Blast.cgi) indicated that G. oxydans 2-ketoreductase aminoacid sequence included a short chain dehydrogenase domain(gnI|Pfam|pfam00106) extending though amino acid positions 4-255. Inaddition, BLAST 2.2.1 analysis (S. F. Altschul et al., 1997, NucleicAcids Res. 25:3389-3402; http://www.ncbi.nlm.nih.gov/BLAST/) indicatedthat the amino acid sequence of the G. oxydans 2-ketoreductase shared53% identity with the amino acid sequences of acetoin reductase andmeso-2-,3-butanediol dehydrogenase from Klebsiella pneumoniae (GenBankAccession Nos. MC78679 and BM13085); 49% identity with the amino acidsequence of L-2,3-butanediol dehydrogenase from Corynebacteriumglutamicum (GenBank Accession No. BAA36159); and 51% identity with theamino acid sequence of a putative short chain oxidoreductase fromStreptomyces coelicolor (GenBank Accession No. T36396).

BLAST 2.2.1 analysis further indicated that longest stretch of identicalcontiguous amino acids shared by G. oxydans 2-ketoreductase and K.pneumoniae acetoin reductase was 9 residues in length. The longeststretch of identical contiguous amino acids shared by G. oxydans2-ketoreductase and K. pneumoniae meso-2-,3-butanediol dehydrogenase was8 residues in length. The longest stretch of identical contiguous aminoacids shared by G. oxydans 2-ketoreductase and C. glutamicumL-2,3-butanediol dehydrogenase was 7 residues in length. The longeststretch of identical contiguous amino acids shared by G. oxydans2-ketoreductase and Streptomyces coelicolor oxidoreductase was 11residues in length.

The G. oxydans 2-ketoreductase nucleotide sequence did not showsignificant homology to previously identified enzymes. BLAST 2.2.1analysis indicated that he longest stretch of identical contiguousnucleotides shared by G. oxydans 2-ketoreductase and other knownnucleotide sequences was 20 bases in length.

Example 5 Subcloning and Expression of G. Oxydans 2-Ketoreductase in E.Coli

To facilitate PCR-based cloning of the 2 ketoreductase gene intoexpression vector pBMS2000 (disclosed in U.S. Pat. No. 6,068,991, issuedMay 30, 2000 to S. W. Liu et al.), oligonucleotide primers containingthe 5′ and 3′ end of the gene along with compatible restrictionendonuclease cleavage sites were prepared to include the followingsequence: 5′ ggaattccatatatccct (5′ end of gene; ttctggaaaatcgc 3′ SEQID NO:9)           NdeI 5′ cgggatcctctcagcgga (3′ end of gene;anti-sense; aaacg 3′ SEQ ID NO:10)           BamHI

High-fidelity amplification of the 2-ketoreductase gene was carried outin four 25 μl aliquots, each consisting of 1× Z-Taq reaction buffer(PanVera Co., Madison, Wis.), 0.2 μM each deoxynucleotide triphosphate(dATP, dCTP, dGTP, dTTP), 0.4 nM each oligonucleotide, 2.5 U Z-Taq DNApolymerase (PanVera), and 10 pg plasmid DNA containing the cloned2-ketoreductase gene. The amplification conditions included incubationat 94° C. for 4 min followed by 25 cycles of incubation at 94° C. for 1min; 50° C. for 1 min; and 72° C. for 1.5 min using a Perkin-Elmer Model480 thermocycler with autoextension. The PCR samples wereelectrophoresed on a 1.0% TAE agarose gel for 2 hr at 100 V. The 780 bpfragment containing the 2-ketoreductase gene was excised from the geland purified using the QIAquick Gel Extraction Kit (QIAGEN).

The concentrations of the isolated DNAs were estimated byelectrophoresis with the Low Molecular Weight DNA Mass Ladder(Invitrogen). Purified DNA was digested with 20 U NdeI for 2 hr at 37°C. in a total volume of 20 μl, diluted to 40 μl with water. This wasfollowed by digestion with 20 U of BamHI at 30° C. for 2 hr. Theexpression vector pBMS2000 was digested with these endonucleases inparallel. The digested samples were electrophoresed on a 1.0% TAEagarose gel for 2 hr at 100 V. The 800 bp and 4516 bp fragmentscontaining the ketoreductase gene and plasmid DNA, respectively, wereexcised from the gel and purified using the QIAquick Gel Extraction Kit(QIAGEN).

The concentrations of the isolated DNAs were estimated byelectrophoresis and comparison with Low Molecular Weight DNA Mass Ladder(Invitrogen). Ligation and transformation were carried out as describedabove. Cells containing plasmid were selected on LB agar containing 20μg/ml neomycin at 37° C. for 20 hr. Plasmids with the desired insertwere screened by colony PCR as described earlier. Neomycin-resistantcolonies were picked using a disposable plastic inoculation needle,swirled into LB broth, and then transferred to LB-neomycin agar. PCRsamples were electrophoresed on a 0.8% TAE agarose gel for 2 hr at 100V. Seven samples out of ten showed a strong band at 800 bp. One colonycontaining this plasmid (named pBMS2000-KR) was chosen for furtherstudy. The cloned G. Oxydans 2-ketoreductase gene was deposited at theAmerican Type Culture Collection (ATCC), 10801 University Boulevard,Manassas, Va. 20110-2209 on Nov. 15, 2001 in E. coli cells as SC16469under ATCC Accession No. PTA-3864 according to the terms of the BudapestTreaty.

The recombinant plasmid was transformed into E. coli strain BL21 (DE3)(Invitrogen, Carlsbad, Calif.) by electroporation. Transformed cellswere selected on LB-neomycin agar medium, and individual colonies wereinoculated into 10 ml MT3 medium (1.0% NZAmine A, 2.0% Yeastamin, 2.0%glycerol, 0.6% Na₂HPO₄, 0.3% KH₂PO₄, 0.125% (NH₄)₂SO₄, and 0.0246%MgSO₄) containing 30 μg/ml neomycin. The cultures were incubated at 28°C. at 250 rpm for 20 hr. Cultures were then diluted in fresh medium,grown to an OD₆₀₀ nm of 0.25, and then incubated under the sameconditions until the OD₆₀₀ reached 0.8±0.1. IPTG was added to a finalconcentration of 0.3 mM and the cultures grown at the above conditionsfor 20 hr. Cells were pelleted by centrifugation (5,000×g) for 7 min.The culture medium was removed, and cells were washed with an equalvolume ice cold 50 mM KPO₄ buffer (pH 7.3) with 2 mM dithiothreitol. Thecells were again pelleted, and the wet cell weight was recorded.

Example 6 Reduction of 2-Pentanone Using Recombinant 2-Ketoreductase

To demonstrate the utility of the recombinant enzyme, the clonedketoreductase was used in the reduction of 2-pentanone to 2-pentanol.The reaction contained 0.18 mg NAD+, 30 mg sodium formate, 0.3 unitsformate dehydrogenase (Sigma, St. Louis, Mo.), 2 mg 2-pentanone, and 0.5ml of extract from an E. coli (BL21(DE3)) culture containing thepBMS2000-KR plasmid and expressing the ketoreductase. Alternatively, P.pastoris formate dehydrogenase (see S. Goldberg et al.: U.S. ProvisionalPatent Application Ser. No. 60/341,934 filed Dec. 19, 2001 under DocketNo. D0170PSP; U.S. Provisional Application Ser. No. 60/375,530 filedApr. 25, 2002 under Docket No. D0170 PSP1; and U.S. Patent ApplicationSer. No. ______ filed concurrently herewith under Docket No. D0170 NP;the contents of which are hereby incorporated by reference in theirentirety) can be substituted for the commercial formate dehydrogenase.Cell extracts were obtained as follows: 2 g recombinant cells wassuspended in 10 ml Buffer A (50 mM Tris-HCl, pH 7.5, 1 mM CaCl₂, and 1mM MgCl₂). The resuspended cells were sonicated for 2 min (20 sec pulse“on” and 30 sec pulse “off”) using Model 550 Sonic Dismembrator (MisonixInc., Farmingdale, N.Y.). The resulting mixture was centrifuged for 15min at 8000 rpm at 4° C. The supernatant was removed and used forreduction reactions. The reactions were carried out in a culture tube at28° C. with shaking at 200 rpm. After 16 hr, samples were quenched with2 ml of ethyl acetate and analyzed by gas chromatography (describedearlier). There was complete reduction of the substrate usingrecombinant enzyme, while no reaction took place in the absence ofrecombinant enzyme.

Example 7 Reduction of Other Alkylketones Using Recombinant2-Ketoreductase

To demonstrate the utility of the recombinant enzyme in the reduction ofother substrates, the cloned ketoreductase was used in the reduction of2-heptanone, 2-octanone, and 2-decanone. The reactions contained 0.18 mgNAD+, 30 mg sodium formate, 0.3 units formate dehydrogenase (purchasedfrom Sigma), 2 mg 2-ketones, and 0.5 ml of extract from the E. coliculture containing the pBMS2000-KR plasmid and expressing theketoreductase (described above). The reactions were carried out in aculture tube at 28° C. on a shaker at 200 rpm. The end of 16 hr, sampleswere quenched with 2 ml of ethyl acetate and analyzed by gaschromatography (described earlier). There was complete reduction of thesubstrates using recombinant enzyme as shown in the following table.Substrate Product % Conversion 2-Heptanone 2-Heptanol >98 2-Octanone2-Octanol >98 2-Decanone 2-Decanol >98

No reaction took place in the absence recombinant enzyme.

The experiments described in Examples 1-7 are also described in S.Goldberg et al.: U.S. Provisional Patent Application Ser. No. 60/341,934filed Dec. 19, 2001 under Docket No. D0170PSP; U.S. ProvisionalApplication Ser. No. 60/375,530 filed Apr. 25, 2002 under Docket No.D0170 PSP1; and U.S. Patent Application Ser. No. ______ filedconcurrently herewith under Docket No. D0170 NP; the contents of whichare hereby incorporated by reference in their entirety.

As various changes can be made in the above compositions and methodswithout departing from the scope and spirit of the invention, it isintended that all subject matter contained in the above description,shown in the accompanying drawings, or defined in the appended claims beinterpreted as illustrative, and not in a limiting sense.

The contents of all patents, patent applications, published articles,books, reference manuals, texts and abstracts cited herein are herebyincorporated by reference in their entirety to more fully describe thestate of the art to which the present invention pertains.

1. (canceled)
 2. (canceled)
 3. (canceled)
 4. (canceled)
 5. (canceled) 6.(canceled)
 7. (canceled)
 8. (canceled)
 9. (canceled)
 10. (canceled) 11.(canceled)
 12. (canceled)
 13. An isolated polypeptide comprising aminoacid sequence SEQ ID NO:2.
 14. An isolated polypeptide comprising theshort chain dehydrogenase domain of amino acid sequence SEQ ID NO:2,wherein said isolated Polypeptide has 2-ketoreductase activity, andwherein the short chain dehydrogenase domain is amino acids 4-255 of SEQID NO:2.
 15. An isolated polypeptide comprising an amino acid sequencethat shares at least 95% sequence identity with the isolated polypeptideof claim 13, wherein said isolated polypeptide has 2-ketoreductaseactivity.
 16. An isolated polypeptide that is a 2-ketoreductase encodedby the nucleotide sequence contained in the plasmid in the ATCC depositdesignated PTA-3864.
 17. An isolated polypeptide of claim 13, whereinsaid isolated polypeptide is recombinant.
 18. (canceled)
 19. (canceled)20. A method of producing a chiral alcohol comprising: incubating theisolated polypeptide of claim 13 with a ketone substrate underconditions to allow reduction of the ketone substrate, thereby producinga chiral alcohol.
 21. The isolated polypeptide of claim 14, wherein saidisolated polypeptide shares at least 95% sequence identity with theamino acid sequence SEQ ID NO:2.
 22. A method of producing a chiralalcohol comprising: incubating the isolated polypeptide of claim 14 witha ketone substrate under conditions to allow reduction of the ketonesubstrate, thereby producing a chiral alcohol.
 23. A method of producinga chiral alcohol comprising: incubating the isolated polypeptide ofclaim 15 with a ketone substrate under conditions to allow reduction ofthe ketone substrate, thereby producing a chiral alcohol.
 24. A methodof producing a chiral alcohol comprising: incubating the isolatedpolypeptide of claim 16 with a ketone substrate under conditions toallow reduction of the ketone substrate, thereby producing a chiralalcohol.
 25. A method of producing a chiral alcohol comprising:incubating the isolated polypeptide of claim 21 with a ketone substrateunder conditions to allow reduction of the ketone substrate, therebyproducing a chiral alcohol.
 26. The method of claim 22, wherein saidketone is selected from the group consisting of 2-pentanone,2-heptanone, 2-octanone, 2-decanone, and 2-hexanone.
 27. The method ofclaim 23, wherein said ketone is selected from the group consisting of2-pentanone, 2-heptanone, 2-octanone, 2-decanone, and 2-hexanone. 28.The method of claim 26, wherein said ketone is 2-pentanone.
 29. Themethod of claim 27, wherein said ketone is 2-pentanone.
 30. An isolatedpolypeptide of claim 14, wherein said isolated polypeptide isrecombinant.
 31. An isolated polypeptide of claim 15, wherein saidisolated polypeptide is recombinant.
 32. An isolated polypeptide ofclaim 16, wherein said isolated polypeptide is recombinant.
 33. Anisolated polypeptide of claim 13, wherein said isolated polypeptideconsists of the amino acid sequence of SEQ ID NO:2.